UMEÅ UNIVERSITY MEDICAL DISSERTATIONS

New series no.1030, ISSN 0346-6612, ISBN 91-7264-089-8

Molecular Genetics of B- and T Lymphocyte

Development

Ingela Wikström

Department of Medical Biosciences

Unit of Medical and Clinical Genetics

Umeå University

Sweden 2006

2

Department of Medical Biosciences

Unit of Medical and Clinical Genetics

Umeå University

SE-901 85 Umeå, Sweden

Copyright © 2006 by Ingela Wikström

ISSN 0346-6612

ISBN 91-7264-089-8

Printed by Solfjädern Offset AB in Umeå, Sweden, 2006

3

Table of contents

Abstract………….……………………………………………………………......................5

Abbreviations………………………………...……………………………………………...6

Articles……………………………………………………………………...……….………7

Introduction…………………………………………………………………………………8

1.1 The immune system…………………………………………...………………………...8

The non-specific immune system………………………………………………………...8

The specific immune system……………………………………………………………..8

1.2 Function of lymphocytes………………………………………………………………..9

B lymphocytes…………………………………………………………………………....9

T lymphocytes…………………………………………………………………………..13

1.3 Lymphocyte development……………………………………………………………..17

B cell development………………………………………………………………….…..17

T cell development……………………………………………………………………...21

2.1 Transcription factors controlling lymphocyte development….…………………….25

2.2 Basic helix-loop-helix transcription factors………………………………………….27

Structure, function and subclasses of bHLH proteins…………………………………..28

Regulation of E-protein activity………………………………………………………...30

E-proteins in B cell development……………………………………………………….31

E-proteins in T cell development………………………………………………………..35

Signalling pathways regulating E-protein function…………………………………..…37

4

Aims of the study…………………………………………………………………….…….38

Results and discussion……………………………………………………………………..38

Paper I: The role of Dµ protein in B cell development…………………………...…38

Paper II: The role of E2-2 in B cell development………………………………...…42

Paper III: The role of E2-2 in T cell development……………………………..……45

Conclusions………………………………………………………………………………...49

Acknowledgements………………………………………………………………………...50

References………………………………………………………………………………….52

Articles and manuscripts………………………………………….....................................73

5

Abstract

Lymphocytes are essential for the generation of specific immunity. Development of B cells in the

bone marrow and T cells in the thymus have several analogous features, and are tightly regulated

processes. Even though there is an increasing amount of information concerning lymphopoiesis,

many questions remain unanswered. The aim of this thesis has been to understand some of the

molecular events that contribute to the control of lymphocyte development.

Expression of the B cell receptor is an important checkpoint in B lymphocyte development.

The Dµ protein is a truncated B cell receptor that can induce some of the signals elicited by full

length µ, but cannot promote further B cell differentiation. In order to determine if this could stem

from an impaired survival signal, we introduced Bcl-2 into RAG2 deficient Dµ transgenic mice.

Analysis of these mice showed that Dµ could not support pre-B cell maturation despite extended

survival of B cell precursors by Bcl-2. In addition, data from recombination competent Dµ

transgenic mice demonstrated that the Dµ induced partial block is permissive for marginal zone B

cell development, whereas the formation of follicular B cells is severely reduced.

The bHLH family of transcription factors is known to be involved in the regulation of

lymphocyte development. Whereas the roles of E2A and HEB have been well documented in both

B- and T-lymphocytes, detailed knowledge concerning E2-2 is lacking. To address the role of E2-2

in B cell development, we have reconstituted mice, using E2-2 deficient fetal liver cells, and

analysed the B cell compartments. We also measured mRNA expression patterns for the three E-

proteins in wildtype mice. Resulting data show that, in addition to a role in B cell lineage entry, E2-

2 is required for efficient expansion of pro-B cells, and also influences the follicular versus

marginal zone decision.

While focusing on assigning a role for E2-2 in T-cell development, we analyzed the

expression of the E-proteins during this process and performed functional studies in fetal thymic

organ cultures. E2-2 deficient mouse embryos were shown to display a partial block at the DN3

stage, which was not due to proliferation or apoptosis defects. In addition, analysis of expression

levels of the pre-Tα chain suggests that E2-2 may play a role in the regulation of transcription of

pre-Tα, and therefore in the assembly of the pre-T cell receptor.

6

Abbreviations

APC antigen presenting cell BCR B cell receptor bHLH basic helix-loop-helix BM bone marrow C constant CTL cytotoxic T lymphocyte D diversity DC dendritic cell DN double negative DP double positive FL fetal liver H heavy Het heterozygous Ig immunoglobulin ISP immature single positive ITAM immunoreceptor tyrosine-based activation motif J joining KO knockout L light MHC major histocompatibility complex MZ marginal zone pBCR pre-B cell receptor pTCR pre-T cell receptor pTα pre-TCRα chain RAG recombination activating gene RF reading frame RSS recombination signal sequence SLC surrogate light chain SP single positive TCR T cell receptor TG transgene TH T helper lymphocyte V variable WT wildtype

7

Articles

I. Wikström I., Bergqvist I., Holmberg D. and Forssell J. (2006) Dµ expression causes

enrichment of MZ B cells, but is non permissive for B cell maturation in Rag2-/- mice even

if combined with Bcl-2. Mol Immunol, 2006 Mar;43(9):1316-24

II. Wikström I.∗, Forssell J.∗, Penha-Goncalves M., Colucci F. and Holmberg D. E2-2

regulates the expansion of pro-B cells and follicular versus MZ decisions. Submitted April

2006

III. Wikström I., Penha-Goncalves M., Forssell J., Bergqvist I. and Holmberg D. A non-

redundant role for the bHLH transcription factor E2-2 in early thymocyte development.

Manuscript under revision in Eur J Immunol

∗ These authors contributed equally to this paper

8

Introduction

1.1 The immune system

Each day we are challenged by potentially disease-causing microorganisms like viruses,

bacteria, fungi and parasites. Luckily our body has developed a system designed to battle

intruders that enter our blood or tissues; the immune system. One fundamental property of

this system is that it can discriminate between self and non-self and therefore clear

infections without causing too much damage to self-tissue. In vertebrates the immune

system consists of both non-specific (innate) and specific (adaptive) components that are

dependent on each other and work in close collaboration to rid the body of pathogens.

The non-specific immune system is also called the innate immune system and is our first

line of defence, immediately able to combat a wide range of pathogens without the need for

prior exposure. The epithelial surfaces of the body are the first defence against infection, not

only providing a physical barrier to infections but also producing antibacterial enzymes and

peptides. Low pH and the normal flora of non-pathogenic bacteria are other obstacles that

intruders must overcome in order to manifest an infection. Innate immunity also involves

phagocytic cells, like macrophages and neutrophils that engulf and destroy microorganisms,

as well as the complement system that recruits inflammatory cells, lyses pathogens or

targets them for phagocytosis by other cells. Only if an infectious organism can break the

early defence set up by the innate immune system will an adaptive immune response

develop.

The specific (adaptive) immune system does not become fully activated until several days

after infection. It develops during the lifetime of an individual as an adaptation to infections

and in many cases it confers lifelong immunity to re-infection from the same pathogen. The

adaptive immune system involves antibodies against a specific pathogen, the humoral

immune response, as well as the cell-mediated immune response, where infected cells are

destroyed by T lymphocytes. These immune responses depend upon the clonal selection of

lymphocytes. Each lymphocyte has a unique antigen receptor, generated by somatic gene

rearrangements, that will scan the surroundings for pathogens. If a lymphocyte encounters

9

the antigen to which its receptor is specific, it will become activated, proliferate, and give

rise to many cells with the same specificity. These antigen-specific effector lymphocytes

will then fight the infection. After the infection has been cleared, some lymphocytes persist,

constituting immunological memory, which ensures a more rapid and effective response

upon a subsequent encounter with the same antigen.

Lymphocytes develop and mature in the primary lymphoid organs; the bone marrow and the

thymus, while activation and accumulation occur in the secondary lymphoid organs; the

lymph nodes and the spleen. The peripheral lymphoid organs are specialized for trapping

antigen, allowing for initiation of immune responses and providing signals that sustain

recirculation of lymphocytes. The whole system is inter-connected by a network of lymph-

and blood vessels that transport lymphocytes and antigens until they encounter one another

in the secondary lymphoid organs.

1.2 Function of lymphocytes

B lymphocytes

The specific immune system consists of B lymphocytes and T lymphocytes, which have

very different ways of recognizing antigen. The antigen receptor of B lymphocytes, the B

cell receptor (BCR), is termed antibody or immunoglobulin (Ig). Each developing

lymphocyte generates a unique antigen receptor, which ensures that one cell will only

recognize one antigen (Burnet, 1976). The enormous diversity in receptor specificity is

generated by somatic rearrangements of a large number of gene segments encoding the

antigen-binding site.

The BCR consists of two identical heavy (H) and two identical light (L) chains joined by

disulfide bonds, giving each antibody two identical antigen-binding sites (Figure 1). The

antigen-binding region varies extensively between different antibodies and it is therefore

known as the variable (V) region. Three segments of particular variability can be identified

in the variable domain of both the heavy and the light chains; they are called hypervariable

10

regions and form the antigen-binding site when brought together in the antibody molecule.

The three hypervariable loops determine antigen specificity by forming a surface

complementary to the antigen, and are more commonly termed complementarity-

determining regions (CDRs) (Wu and Kabat, 1970; Amit et al., 1985; Amit et al., 1986).

The region that carries out the effector function does not vary as much and is thus known as

the constant (C) region (Figure 1). There are five main forms or so called isotypes; IgA,

IgD, IgG, IgE and IgM, each with a specialized effector mechanism (Edelman, 1973; Vitetta

and Uhr, 1975).

Figure 1. A schematic representation of the antibody molecule. White boxes represent the

variable antigen-binding domains. Grey boxes represent the constant domains of the heavy

and light chains.

Membrane-bound immunoglobulin on the B cell surface serves as the cell’s receptor for

antigen, while immunoglobulin of the same specificity is secreted as antibody by terminally

differentiated B cells called plasma cells. Secreted antibodies bind pathogens or their toxic

products and constitute the main effector function of B cells in adaptive immunity.

Antibodies can participate in host defence in three main ways. Firstly they can neutralize

toxins and viral particles by coating them and thereby inhibiting their binding to host cells.

Secondly, coating of pathogens and foreign particles by antibodies renders them more easily

recognised and ingested by phagocytes (opsonization). The third function is to activate the

complement system, which eventually eliminates the pathogens by lysis or phagocytosis.

11

When membrane bound, the antibody is associated with the invariant accessory molecules

Igα and Igβ, making up the BCR complex (Hombach et al., 1990). The antibody itself

cannot generate a signal, instead it is the cytoplasmic tails of Igα and Igβ that carry out the

signalling function of the receptor (Sanchez et al., 1993). A motif termed the

immunoreceptor tyrosine-based activation motif (ITAM) is present on each of the Igα and

Igβ tails and signal transduction is initiated when these are phosphorylated (Reth, 1989). A

signalling cascade involving many signalling molecules follows ITAM-phosphorylation.

Ultimately, antigen recognition leads to the induction of new gene synthesis by activation of

transcription factors (discussed later).

In the periphery, B cells require expression of their BCR for survival (Lam et al., 1997). In

addition, the TNF family ligand, BAFF (also called BlyS) is specifically required for the

survival of mature re-circulating B cells (Gross et al., 2001; Schiemann et al., 2001). Upon

binding of foreign antigen to the antibody on its surface, the B cell responds by proliferation

and differentiation. It can either differentiate into an antibody secreting plasma cell or into a

long-term surviving memory cell (Rajewsky, 1996). The B cells can respond to antigens

that are either thymus-dependent (TD) or thymus-independent (TI). TD antigens are soluble

proteins that antigen presenting cells (APCs) can internalize, process and present on their

surface together with class II major histocompatibility complex (MHC) molecules. Peptides

bound to MHC class II molecules trigger helper T cells to make membrane-bound and

secreted molecules, such as cytokines, that can activate a B cell. Many microbial

constituents such as bacterial polysaccharides can induce antibody production in the

absence of T helper cells. These are known as TI antigens, but in contrast to TD responses,

TI responses cannot produce memory cells.

The Toll-like receptors (TLRs) recognize TI antigens, including pathogen associated

molecular patterns (PAMPs), such as bacterial lipopolysaccharides (LPS), RNAs and

DNAs, which are widely recognized by cells from both the adaptive and innate lineages.

The TLRs function in B cells is to induce and/or co-stimulate proliferation, class switching

and differentiation into antibody-secreting cells. B cells are recognized for their expression

12

of TLR9 and TLR10, which are induced in response to BCR stimulation and predominate in

activated and memory populations (Bernasconi et al., 2003; Bourke et al., 2003). TLR9 and

its ligand, CpG-containing DNA, have received a lot of attention due to their nature as a

pathogenic co-stimulator of auto-reactive B cell responses (Leadbetter et al., 2002; Viglianti

et al., 2003).

After T cell-dependent activation, B cells migrate to lymphoid follicles and proliferate to

form germinal centres. The reactions that take place here provide for a more effective

response upon re-infection. For example, every B cell begins by expressing IgM as its BCR

and it is the first antibody produced, later in the immune response, the same V-region may

be expressed in IgG, IgA, or IgE antibodies. This is called isotype switching (Nossal et al.,

1964; Kataoka et al., 1980) and it generates antibodies with different biological functions

but with the same specificity of the receptor. The B cells in the germinal centre also go

through a process called affinity maturation when they undergo V-region somatic

hypermutation (Weigert et al., 1970; Bernard et al., 1978) and cells with improved affinity

for the antigen are selected. Ligation of the BCR and stimulation through CD40, together

with direct T cell contact, are required for both the initiation and maintenance of germinal

centres (Kawabe et al., 1994; Han et al., 1995).

Once an immature B cell expresses IgM on its surface its fate is determined by the

specificity of the antigen receptor. Lymphocytes with strongly self-reactive receptors should

be eliminated to prevent autoimmunity. Immature B cells that bind self-antigens in the bone

marrow are deleted from the repertoire (clonal deletion). These B cells either die through

apoptosis or undergo further rearrangements of the variable part of the light-chain (receptor

editing), alternatively they are rendered anergic (permanent state of unresponsiveness).

These processes are referred to as central tolerance. Self-reactive B cells that manage to

escape this control and migrate from the bone marrow are controlled by peripheral

tolerance mechanisms. For example, antigen recognition by B cells without help from T

cells can make the B cells anergic. In addition, the antigen-induced loss of B cells known as

clonal deletion can also occur in the periphery (Goodnow, 1996; Van Parijs and Abbas,

1998).

13

Newly formed B cells give rise to follicular or marginal zone (MZ) B cells (Kumararatne

and MacLennan, 1981; Martin and Kearney, 2002) that differ in their surface phenotype,

anatomic localization and immunological function. Follicular B cells make up the bulk of

the peripheral B cells, and are the main constituents of the splenic and lymph node follicles.

They recirculate through the blood and lymphatic systems and contribute to both the

primary and memory responses. In contrast, MZ B cells comprise only about 5% of all

peripheral B cells, are localized in the marginal sinus of the spleen, do not recirculate and

contribute to the early primary immune response. As they constitute the first line of defence

against blood-borne pathogens, without contributing to the memory B cell response, they

may be viewed as a link between the innate and adaptive immune systems.

Another subset of B cells, resembling the MZ cells, are the B-1 cells (comprising about 5%

of total B cells). They are distinguished from conventional B cells (B-2) by the expression

of cell-surface molecules, B-1 cells are B220loIgMhiIgD-CD21-CD23-, a fraction of these are

CD5+ (B1a cells), while another subset is CD5- (B1b cells) (Berland and Wortis, 2002). The

B-1 cells have properties quite distinct from conventional B cells, they arise early in

ontogeny and use a distinct and limited set of gene rearrangements to make their receptors.

They are self-renewing in the periphery and reside mainly in the peritoneal cavity

(Hayakawa et al., 1983; Hayakawa et al., 1986; Berland and Wortis, 2002). The B-1 cells

seem to be important in T cell independent responses to carbohydrate antigens such as

bacterial polysaccharides.

T lymphocytes

All T lymphocytes express a T cell receptor (TCR) on their cell surface (Hedrick et al.,

1984; Yanagi et al., 1984). This consists of two chains, the α chain and the β chain, that

together form a heterodimer with a constant region and a variable region, which determines

the specificity of the receptor. Similar to the receptors for B cells, the antigen specificities of

the TCRs are generated through differential usage of variable region gene segments (Siu et

al., 1984). The α:β heterodimer is associated with a complex of signalling chains

collectively called CD3 (Samelson et al., 1985). These invariant accessory chains are; one

14

CD3γ, one CD3δ and two CD3ε, which all contain cytoplasmic ITAM motifs involved in

signal transduction. Consequently, the CD3 complex is required for the surface expression

of the antigen-binding chains and for signalling via the receptor. Optimal expression and

signalling, however, also require a homodimer of ζ chains.

T lymphocytes can first be divided into two groups depending on what co-receptor they

express in addition to the TCR. Cytotoxic T cells (CTL) express CD8 and are specialised to

kill any cell they specifically recognise. The other type of T cells, T helper cells (TH),

express CD4 and their main function is to activate other effector cells of the immune

system. T helper cells can be classified further into two distinct subsets based on their

cytokine production (Mosmann et al., 1986). T helper 1 (TH1) cells produce IFN-γ, IL-2 and

TNF and mediate elimination of intracellular pathogens. T helper 2 (TH2) cells produce IL-

4, IL-5, IL-13 and IL-6 and mediate the clearance of large, extracellular pathogens, as well

as being involved in allergy reactions.

Another category of T helper cells is the T regulatory (Treg) cells, which play a key role in

the maintenance of self-tolerance and negative control of various immune responses.

CD4+CD25+ T cells have been shown to inhibit autoimmune diabetes in mice and rats

(Salomon et al., 2000; Stephens and Mason, 2000) and the transfer of T cells lacking the

CD4+CD25+ subset into athymic nude mice have been shown to result in the spontaneous

development of T cell mediated diseases (Sakaguchi et al., 1995). There are at least two

types of Treg cells: developmentally programmed and inducible. The developmentally

programmed Tregs are derived from the thymus as a functionally mature population. They

are typically characterized by the expression of CD4 and CD25 and their development and

function depends on the transcription factor Foxp3 (Sakaguchi et al., 2001; Fontenot et al.,

2003; Hori et al., 2003; Khattri et al., 2003). Naïve CD4 T cells can be induced by

numerous environmental factors to become regulatory T cells, these inducible Tregs are not

clearly defined in terms of their phenotype and function, but are thought to mediate

suppression predominantly via cytokine-dependent pathways, in contrast to thymically

derived Tregs which require cell-cell contact in order to mediate suppression (Berthelot and

Maugars, 2004).

15

Unlike the BCR that can recognize free antigen, the TCR only recognize antigens coupled

to MHC molecules on the cell surface (Katz et al., 1973; Zinkernagel and Doherty, 1974).

The MHC is polygenic, it contains several different MHC class I and class II genes so that

an individual can have a set of MHC molecules with different peptide-specificities. The

MHC is also highly polymorphic, so there are several variants of each gene within the

population. These two properties make it difficult for pathogens to escape immune

responses mediated in this way. The mouse MHC locus is called H-2 and contains clusters

of MHC class I genes and MHC class II genes. Class I molecules are encoded by the K, D

and L regions and consist of an α-chain associated to the β2-microglobulin molecule. They

are expressed on all nucleated cells. The class II molecules are encoded by the A and E

regions and are made up of αβ-heterodimers. MHC class II molecules are exclusively

expressed by APCs such as B cells, dendritic cells (DCs) and macrophages (Steinmetz et al.,

1982; Kaufman et al., 1984).

DCs provide two signals required for activation of naïve T cells. In addition to the antigen-

specific binding of the TCR to peptide-MHC, a second signal is provided by co-stimulatory

molecules such as B7-1 (CD80) and B7-2 (CD86), expressed by the DCs. Depending on the

density of peptides presented, types of co-stimulatory molecules expressed and cytokines

secreted by the DCs, CD4 T cells differentiate into either TH1 or TH2 cells (Constant and

Bottomly, 1997). CD8 T cells require similar signals from the DCs for activation. The main

pathway by which DCs become activated and mature to provide the second signal to naïve

T cells occurs via the TLR recognition of PAMPs, discussed earlier (Medzhitov, 2001).

Pathogens and their products can be found in either the cytosol or the vesicular

compartment of cells. MHC class I molecules deliver peptides from the cytosol to the cell

surface. Such peptides are usually from degraded endogenous intracellular proteins, but if a

cell is infected by a virus, viral peptides will also be shown on MHC class I molecules and

the cell is targeted for destruction (Townsend et al., 1985; Morrison et al., 1986; Bjorkman

et al., 1987). CTLs recognize peptides associated with MHC class I, the CD8 co-receptor

binds to the constant part of the MHC and strengthens the interaction of the TCR/peptide-

16

MHC (Norment et al., 1988). When the CTL and an infected target cell are in close contact,

the activated CTL releases cytotoxic proteins such as perforin and granzymes in a

directional manner in order to lyse the target cell.

Peptides associated with MHC class II molecules are from exogenous proteins that have

been internalized by phagocytosis or endocytosis (Davidson et al., 1991; Germain and

Hendrix, 1991). The internalized proteins are degraded in specialized vesicles by enzymes

and low pH before binding to MHC molecules and transportation to the cell surface. T

helper cells recognize MHC class II associated peptides and the co-receptor CD4 functions

in a similar way to CD8/MHC class I (Doyle and Strominger, 1987). This interaction leads

to secretion of various cytokines by the T cells, which results in activation of CTL and B

cells.

A small population of T cells (2-5%) carry an alternative receptor made up of γ and δ chains

(Bank et al., 1986; Brenner et al., 1986). The receptor repertoire is limited and it is possible

that it is not restricted by the MHC class I and class II molecules, but may bind free antigen.

Little is known about the biological function of these cells, but they predominate in the

epithelium of the skin and intestines.

When T cells mature in the thymus they go through a series of selection events to ensure

that no auto-reactive cells develop (described later). Even so, some self-reactive T cells

escape to the periphery where they are under the control of peripheral tolerance

mechanisms much like those for B cells. For example, T cells may be rendered anergic if

antigen recognition occurs without co-stimulation from APCs. T cells might also be

eliminated by Fas-induced apoptosis when repeatedly activated by persistent antigens such

as self-antigen (Goodnow, 1996; Van Parijs and Abbas, 1998).

17

1.3 Lymphocyte development

B cell development

All hematopoietic cells, including B and T lymphocytes, are derived from pluripotent stem

cells (Abramson et al., 1977). Hematopoiesis can first be detected outside the embryo in the

yolk sac, it then proceeds in the fetal liver and after birth the bone marrow (BM) becomes

the permanent site of hematopoiesis (Osmond and Nossal, 1974; Raff et al., 1976).

The development of B cells occurs in the BM and is independent of foreign antigen.

Survival of early B cells is dependent upon direct contact with stroma and growth factors, of

which IL-7 is one of the most critical (Namen et al., 1988; Hardy et al., 1991; Peschon et al.,

1994; von Freeden-Jeffry et al., 1995).

The process of B cell development can be divided into several stages based on changes in Ig

gene rearrangement status and expression of specific protein markers. The classifications

according to Hardy and Rolink, are summarized in Figure 2 (Hardy et al., 1991; Rolink et

al., 1994). During differentiation, the variable regions of the antibody´s H and L chains are

assembled from a pool of Ig germ-line gene segments, called variable (V), diversity (D) and

joining (J) gene segments. The rearrangement process is mediated by recombination signal

sequences (RSS) flanking the gene segments (Early et al., 1980; Sakano et al., 1980).

Proteins encoded by the recombination activating genes (RAG-1 and RAG-2) recognise the

RSSs and initiate the rearrangement process (Schatz et al., 1989; Oettinger et al., 1990) by

creating double-stranded breaks followed by repair mediated by general repair enzymes

(Smider and Chu, 1997). The absence of either RAG gene completely abrogates

rearrangement and results in individuals lacking B and T cells (Mombaerts et al., 1992b;

Shinkai et al., 1993).

18

Figure 2. A schematic diagram of B cell differentiation in the BM, showing the different

developmental stages. The expression of B-lineage associated genes and the configuration

of Ig genes are indicated. The nomenclatures according to Hardy et al. (1991) and Rolink et

al. (1994) are shown together with expression of markers used to define the stages. B220 is

expressed at all stages. The black arrow indicates the stage at which B cell development is

blocked upon failure to express the pBCR.

BCR rearrangements start at the pro-B cell stage, where the heavy chain is rearranged first.

One of the 15 D gene segments (Kurosawa and Tonegawa, 1982) is joined to one of four JH

gene segments (Sakano et al., 1980), and then one of several hundred VH gene segments

(Brodeur and Riblet, 1984; Livant et al., 1986) is joined to the DJH complex at one allele. If

the join is non-productive (out-of-frame), the second allele rearranges a VH to the DJ-

complex. The VHDJH complex is thereafter attached to the constant part of the Ig through

mRNA splicing. After a functional VHDJH rearrangement has been made, the µ heavy chain

is expressed on the cell surface, together with the surrogate light chain (SLC) (Sakaguchi

and Melchers, 1986; Kudo and Melchers, 1987; Tsubata and Reth, 1990). The SLC is

composed of two polypeptides, VpreB and λ5. There are two VpreB genes in the mouse,

VpreB1 and VpreB2 (Sakaguchi and Melchers, 1986; Kudo and Melchers, 1987) both of

19

which can associate with λ5 to form the SLC (Karasuyama et al., 1990; Dul et al., 1996).

Mice lacking any SLC components show an incomplete block in B cell development

(Kitamura et al., 1992; Mundt et al., 2001; Shimizu et al., 2002).

The µ/SLC complex is termed the pre-B cell receptor (pBCR) and it is believed to, in

association with Igα and Igβ, mediate a signal to stop further rearrangements at the heavy

chain locus (allelic exclusion) (Nussenzweig et al., 1987; Manz et al., 1988). Allelic

exclusion of the IgH locus might also be achieved through an alternative pathway that does

not require pBCR function, as indicated by mice that show allelic exclusion despite lacking

the SLC (Shimizu et al., 2002). The pBCR also signals to up regulate light chain

rearrangement (Iglesias et al., 1991; Shapiro et al., 1993), induce silencing of VpreB and λ5

transcription (Parker et al., 2005) and to continue differentiation to the pre-B cell stage

(Kitamura et al., 1991).

In addition to providing the checkpoint that monitors functional IgH recombination, the

pBCR also signals for proliferation of pre-B cells (Melchers, 2005). While the expansion at

the pro-B cell stage has been shown to be dependent on the IL7-Rα chain (Peschon et al.,

1994; von Freeden-Jeffry et al., 1995; Maki et al., 1996; Maraskovsky et al., 1998; Carvalho

et al., 2001; Sitnicka et al., 2003b), optimal cell expansion at the pre-B cell stage is

dependent on both the expression of pBCR and IL7-Rα (Erlandsson et al., 2005). The fact

that pre-B cells go through several rounds of division before rearrangement of the light

chain (Decker et al., 1991) increases the repertoire diversity by permitting one functional

heavy chain to pair with a wide range of light chains. However, rearranged heavy chains are

not expanded equally, since there is a skewing towards increased representation of distal VH

gene families. This may be due to better pairing of these rearrangements with SLCs and

hence increased proliferation (Martensson et al., 2002).

The light chain gene rearrangement is analogous to the heavy chain. The κ light-chain locus

tends to rearrange before the λ locus by joining a Vκ to a Jκ gene segment. As with the heavy

chain locus, rearrangements take place at only one allele at a time. Only if the first

20

rearrangement is non-productive, the second allele is rearranged. This mechanism, called

allelic exclusion, ensures that each cell expresses immunoglobulins with a single antigen

specificity (Yancopoulos and Alt, 1986). The mechanism of this process is not fully

understood, however, it has been proposed that chromosomal accessibility is a primary

regulator of V-DJ recombination (Sleckman et al., 1998). After the generation of a light

chain, immature B cells express complete BCRs that consist of a light chain and a heavy

chain. If this surface IgM is self-reactive, the cell may change the BCR-specificity by nested

light chain gene rearrangements, receptor editing, (Tiegs et al., 1993; Radic and Zouali,

1996) or it will die by antigen-induced apoptosis (Hartley et al., 1993; Melamed et al.,

1998).

The last stage in the BM involves the co-expression of the δ chain and emigration to the

periphery, leading to the generation of mature IgM/IgD positive cells. Upon encounter with

foreign antigen, the antigen-dependent phase of B cell differentiation begins. This involves

proliferation and differentiation into either antibody secreting plasma cells or long-lived

memory cells.

The importance of the pBCR and the BCR in B cell development has been demonstrated

when transgenic µ-H chains and H chains together with L chains are introduced into RAG

deficient mice (Spanopoulou et al., 1994; Young et al., 1994). When a transgenic µ-H chain

is introduced, B cell development can proceed until the pre-B cell stage, where the L chains

normally rearrange, but then stops. If both transgenic H chains and L chains are introduced,

B cell development is completely rescued.

Assembly of the heavy and light chains from rearrangement of a pool of gene segments

generates enormous diversity in antibody specificity. Another type of combinatorial

diversity is generated by the combination of the two different receptor chains, the heavy

and the light chain. In addition, other mechanisms increase the diversity by exonuclease

nibbling, addition of P-nucleotides and insertion of N-sequences, catalyzed by terminal

deoxynucleotidyl transferase (TdT) (Alt and Baltimore, 1982).

21

The utilization of VH genes differs in fetal and adult life. B cells that are produced during

fetal and neonatal life preferentially use D-proximal VH genes, while the repertoire in adult

mice shows random VH gene utilization (Yancopoulos et al., 1984; Perlmutter et al., 1985).

Selection at the cellular level is likely to contribute to the establishment of the mature B cell

repertoire. Antibodies produced early in development are also characterized by lack of N-

sequence additions due to the absence of TdT activity at this time (Carlsson and Holmberg,

1990; Feeney, 1990; Gu et al., 1990).

During B cell development in mice, a truncated µ chain can be formed before the

rearrangements at the heavy chain locus are completed. The majority of DH segments have

their own promoter and an ATG translational initiation codon, so that when DH is joined to

JH in one of three possible reading frames, reading frame 2 (RF2) (Ichihara et al., 1989), the

resulting DHJH complex can be translated into a truncated µ chain denoted Dµ (Reth and

Alt, 1984). Joints in RF2 are shown to be underrepresented in pre-B or mature B cells

(Meek, 1990; Gu et al., 1991; Carlsson et al., 1992), suggesting that Dµ expression blocks

further differentiation (Gu et al., 1991; Chang et al., 1992; Ehlich et al., 1994; Haasner et

al., 1994). Indeed, expression of a Dµ transgene results in a partial block in B cell

development at the pro-B cell stage, with suppression of endogenous µ chain gene

rearrangements (Tornberg et al., 1998; Malynn et al., 2002).

T cell development

T cells are derived from stem cells in the bone marrow, but migrate at an early stage to the

thymus where differentiation and TCR gene rearrangement occur. The rearrangement is

essentially similar to VDJ rearrangement in B cells except that T cells have two different

sets of receptors, αβ and γδ. The development of T cells from a multipotent progenitor is a

complex process that is dependent on genetic recombination and extracellular signals.

Proliferation and survival of early thymocyte progenitors depends largely on signals from

the IL-7-IL-7R and the tyrosine kinase receptor c-kit and its ligand, stromal cell factor

(SCF). During later stages of development, thymocytes depend upon cytokines and signals

through the pre-TCR and TCR (Cantrell, 2002).

22

Distinct developmental stages of progenitor T cells can be defined by the expression of the

accessory molecules CD4 and CD8 (Godfrey et al., 1993). The most immature thymocytes

express neither CD4 nor CD8 (double-negative stage, DN) and further development leads,

after a stage as immature CD8 single–positive (ISP), to the expression of both CD4 and

CD8 (double-positive stage, DP). The DP cells then mature into fully differentiated cells

that express either CD4 or CD8 (CD4 or CD8 single-positive stage, SP) and leave the

thymus (Haks et al., 1999; von Boehmer et al., 1999). The surface proteins CD25 (IL-2

receptor α-chain), CD44 and c-kit (CD117) can be used to further subdivide the DN cells

into four additional developmental stages, DN1 to DN4 (Godfrey et al., 1992; Godfrey et

al., 1993; Hoffman et al., 1996). A schematic overview of T cell development is presented

in Figure 3.

Figure 3. The different stages of T cell development in the thymus. Surface markers defining

the stages are shown. The black arrow indicates where β-selection takes place while the

white arrow shows the stage where positive and negative selection occurs.

The DN1 population (CD44+, CD25-, c-kit+) consists of the earliest T cell precursors in the

thymus. These are not yet fully committed to the T cell lineage but could also differentiate

into NK or DCs. However, they can no longer become myeloid or erythroid lineage cells

(Wu et al., 1991; Ardavin et al., 1993). Successful rearrangement of the β chain of the TCR

gene at the transition to the DN3 stage (CD44-, CD25+) leads to the assembly and

23

expression of a pTCR that contains the rearranged β chain and the surrogate α-chain,

denoted pTα, together with signalling molecules of the CD3 complex (Groettrup et al.,

1993; Saint-Ruf et al., 1994).

Expression of the pTCR on the cell surface enables the cell to go through an important

checkpoint called β-selection (Mallick et al., 1993; Dudley et al., 1994). Thymocytes

belonging to the CD4/CD8 double-negative (DN) stage are critically dependent on pTCR

signalling for survival, proliferation and developmental progression to the more mature

CD4/CD8 double-positive (DP) stage (Chaffin et al., 1990; von Boehmer et al., 1999). In

mice unable to produce β-chains, T cell development is blocked at the DN3 stage

(Mombaerts et al., 1992a; Mombaerts et al., 1992b; Shinkai et al., 1992). In RAG-/- mice,

this block can be overcome by introduction of a rearranged TCR β-chain transgene or by the

injection of anti-CD3ε antibodies that can drive thymocyte differentiation to the DP stage

(Levelt et al., 1993; Shinkai et al., 1993). Similar blocks at the DN3 stage occurs in pTα,

CD3ε and p56lck deficient mice, demonstrating the importance of a signalling competent

pTCR (Molina et al., 1992; Fehling et al., 1995; Malissen et al., 1995).

The rearrangement of β-, γ-, and δ-chain genes occurs almost simultaneously at the DN

stage, at this time the thymocytes are not yet committed to the αβ or γδ lineages. When the

T cell expresses pTCR on its surface, it becomes committed to the αβ lineage and further

rearrangements at the γδ locus cease. The mechanisms controlling this branch point are not

clear, but signalling through the receptors of the Notch family might be involved in the

lineage decision, although experiments have given contradictory data regarding this

(Washburn et al., 1997; Kang et al., 2001; Wolfer et al., 2002).

After passing β-selection, thymocytes undergo proliferation and differentiate to the DP

stage. This transition is characterized by down regulation of CD25, induction of CD4 and

CD8 expression and cell division. When the cells reach the DP stage they become small and

cycle slowly, RAG expression is up regulated and now the rearrangement of the α-chain

takes place (Wilson et al., 1994). The α-chain then pairs with the β-chain to make up the

24

complete TCR, enabling the cell to go through the checkpoints called positive and negative

selection (see next paragraph). Cells surviving the selection at the DP stage up regulate the

expression of TCR and become SP TCRhi cells. They leave the thymus and enter the

peripheral pool of naïve mature T lymphocytes.

To be able to generate an immune response, T cells are first screened for recognition of self-

MHC molecules, a process known as positive selection (von Boehmer and Kisielow, 1990).

Those T cells that fail to bind to the MHC molecules die (death by neglect) (Jameson and

Bevan, 1998). Thymocytes that recognise MHC class I lose the expression of CD4 while

those that recognise MHC class II lose expression of CD8 to become SP T cells.

Thymocytes recognising self-MHC or self-peptides plus self-MHC with too high affinity

are deleted through negative selection (Kisielow et al., 1988). These two mechanisms

ensure that T cells that go to the periphery respond only to foreign antigen plus self-MHC

and that self-reactive T cells are eliminated. The net result is that most thymocytes are

eliminated during development (up to 99%) and only a few of them are able to reach the

mature stage.

Although MHC specificity in positive selection is the major determinant in the CD4/CD8

lineage decision, several studies support the view that quantitative differences in TCR

signalling in DP thymocytes leads to an instructive bias in CD4/CD8 lineage commitment.

This “strength of signal” model proposes that stronger/longer signals promote the CD4 T

cell lineage and weaker/shorter signals favour the CD8 T cell lineage (Matechak et al.,

1996; Bosselut et al., 2001; Lucas et al., 2002; Pearce et al., 2004; Tyznik et al., 2004).

There also seems to be a requirement for continuous TCR signalling as a re-enforcement

step, even after CD4 or CD8 down regulation (Salmon et al., 1999; Liu et al., 2003). A

variation of the “strength of signal” model is the “kinetic signalling” model, that states that

all thymocytes sensing a TCR signal proceed to a CD4+CD8lo stage (Bosselut et al., 2003).

At this stage, sustained TCR signals lead to CD4 T cell commitment but CD8 T cell

commitment results from the cessation of TCR signals (Liu et al., 2003; Liu and Bosselut,

2004).

25

2.1 Transcription factors controlling lymphocyte development

The development of hematopoietic lineages, including lymphocytes, is a very complex

process. Targeting experiments have identified several different transcription factors

involved in development (Henderson and Calame, 1998; Kuo and Leiden, 1999). Distinct

transcription factor expression patterns serve as hallmarks for each hematopoietic cell type

and the combination of factors used in each pathway can explain the effector genes that the

cell type will express. As illustrated in Figure 4, the cells are passed stepwise from the

control of one set of regulators to another as they progress through development.

Transcription factors SCL/tal-1 and Lmo2 have been shown to be essential for

hematopoietic development (Porcher et al., 1996; Yamada et al., 1998). After commitment

to the lymphoid lineage, other transcription factors are required, some of which are shown

in Figure 4.

The PU.1, a transcription factor of the Ets family, is required for the development of several

cell lineages. PU.1 deficient mice lack lymphoid and myeloid precursors at birth (Scott et

al., 1994). T cells and neutrophils appear at reduced levels after birth while B cells and

macrophages remain absent, indicating that PU.1 is not essential for lymphoid commitment

but for normal differentiation of B cells (McKercher et al., 1996). Constitutive expression of

the IL-7 receptor is sufficient to rescue B cell development from the PU.1 deficient block,

suggesting that the IL-7 signal is downstream of PU.1 (DeKoter et al., 2002).

The Ikaros family of zink-finger transcription factors are required for all lymphocyte

lineages (Georgopoulos et al., 1994). Ikaros mutant mice lack fetal B and T cells while T

cells are generated in adult mice, indicating different functions for Ikaros in fetal and adult

T cell development (Wang et al., 1996). So far only a few genes, such as those encoding

TDT (Trinh et al., 2001), λ5 (Sabbattini et al., 2001), and CD8α (Harker et al., 2002) have

been identified as direct targets of Ikaros.

The basic helix-loop-helix (bHLH) transcription factors, E2A, HEB and E2-2 are also

essential for both B and T cell development and will be discussed in detail later.

26

Figure 4. Selected transcription factors regulating lymphocyte development. *Discussed in

section 2.2 and papers II and III.

The B cell precursor is first rendered competent by its expression of PU.1 and Ikaros,

without which B cell development does not begin (DeKoter et al., 2002; Allman et al.,

2003; Rosenbauer et al., 2004). B cell development is then guided by three main

transcription factors, E2A, EBF (early B cell factor) and Pax5 (paired box protein 5). EBF is

a regulator of early B cell development and mutant mice show a developmental block at the

pro-B cell stage, before any D-J rearrangements (Lin and Grosschedl, 1995). B cell

development in Pax5 deficient mice is arrested at the pro-B cell stage after IgH DJ

rearrangements (Urbanek et al., 1994; Schilham et al., 1996; Hesslein et al., 2003),

subsequent to the block in E2A and EBF mutant mice. Pro-B cells can develop into B cells

in the presence of Pax5, whereas in its absence they can enter other differentiation

pathways. Pax5-/- pro-B cells can reconstitute T cell development in Rag2-/- mice and they

can differentiate into macrophages, osteoclasts, DCs, granulocytes and NK cells when

stimulated in vitro (Nutt et al., 1999; Rolink et al., 1999). One explanation for this is that the

role of Pax5 is to suppress alternative lineage choices.

The complicated differentiation process of T cells requires a strict control of transcriptional

activation and repression and several transcription factors are known to play important roles

(Kuo and Leiden, 1999). Among these are the basic helix-loop-helix (bHLH) proteins

(Massari and Murre, 2000). The first stages of T cell development and expression of T cell

genes depend on the transcription factors GATA-3, c-Myb, members of the Runx family, of

27

the bHLH family and of the Ikaros family. The appearance of pre-T cells in fetal life also

depends on the Ets-family transcription factor PU.1. The one transcription factor that

appears to be T cell-specific is GATA-3. During T cell development, the functions that

these factors perform are discontinuous, stage-specific and even subject to alterations

between activating and inhibitory effects from one stage to the next.

Disruption of the genes encoding GATA-3, HES-1 and c-Myb leads to a complete arrest at

the DN stage and a failure of null mutated ES cells to differentiate into mature T cells in

Rag2-/- complementation assays (Ting et al., 1996; Allen et al., 1999; Tomita et al., 1999).

Transcription factors regulating T cell development in later stages are T cell-factor 1 (TCF-

1) and lymphoid-enhancer-binding factor 1 (LEF-1). Mice that are double mutant for these

factors show a partial block at the DN stage and a complete block at the DN to DP transition

(Okamura et al., 1998).

In the periphery, a large number of transcription factors function in regulating lymphocyte

activation, function and terminal differentiation. Some of these are the Ets, NF-κB/Rel, Oct-

2, OCA-B, NFAT and LKLF proteins (Henderson and Calame, 1998; Kuo and Leiden,

1999).

2.2 Basic helix-loop-helix transcription factors

The bHLH proteins are dimeric transcription factors that are found in eukaryotes ranging

from yeasts to humans (Massari and Murre, 2000; Ledent and Vervoort, 2001). They have

important functions in embryonic development, especially in neurogenesis, myogenesis,

heart development and hematopoiesis (Littlewood, 1998). The bHLH family of transcription

factors was first defined in 1989 when Murre and colleagues identified E12 and E47 by

their ability to bind as homodimers to the E-box sites located within the Ig heavy and Ig

light chain gene enhancers (Murre et al., 1989a; Murre et al., 1989b). After this initial

classification of a few bHLH proteins, many more proteins have been identified and today

the bHLH family contains over 400 different members (Morgenstern and Atchley, 1999).

28

Structure, function and subclasses of bHLH proteins

Members of the bHLH family have two highly conserved and functionally distinct domains,

which together make up a region of approximately 60 amino-acid residues. The HLH motif

consists of two amphipatic α-helices separated by a loop structure (Murre et al., 1989b).

This domain facilitates the dimerization with other HLH members (Murre et al., 1989b). At

the amino-terminal end is a conserved basic region that allows HLH dimers to bind to DNA.

In addition, E-proteins contain two conserved regions that function to recruit coactivator or

corepressor complexes, these are referred to as AD1 and AD2 domains (Aronheim et al.,

1993; Quong et al., 1993; Massari et al., 1999) (Figure 5).

Figure 5. Schematic representation of the human E2-2 protein. The location of the

transactivation, bHLH and C domains are shown.

The DNA binding sites are so called E-boxes, with the consensus sequence CANNTG. This

sequence was first discovered in the Ig heavy chain enhancer (Ephrussi et al., 1985) but is

now found in regulatory elements of numerous genes, such as the TCR α and β loci, the

promoters of mb-1, λ5, pTα and the CD4 silencer and enhancer elements (Ho et al., 1989;

Staudt and Lenardo, 1991; Sawada and Littman, 1993; Ernst and Smale, 1995; Reizis and

Leder, 1999; Kee and Murre, 2001).

Based on the expression patterns and dimerization specificities of the bHLH proteins, they

can be divided into five different classes.

Class I bHLH proteins are also called E-proteins, these are widely, but not ubiquitously

expressed. There are four E-proteins in mammals; these are E47 and E12, in addition to

HEB (HeLa E-box-binding protein) and E2-2 (Murre et al., 1989a; Henthorn et al., 1990;

29

Corneliussen et al., 1991; Sun and Baltimore, 1991; Hu et al., 1992). The Drosophila

melanogaster homolog of E-proteins, “daughterless”, is involved in sex determination and

neurogenesis.

E12 and E47 are encoded by one gene, E2A (also known as Tcfe2a in mice) and arise

through differential splicing (Murre et al., 1989a; Sun and Baltimore, 1991). These two

proteins bind to E-box sites with different affinities; E47 with relatively high affinity,

whereas E12 interacts weakly (Sun and Baltimore, 1991). HEB and E2-2 are encoded by

separate genes (Henthorn et al., 1990; Corneliussen et al., 1991; Hu et al., 1992), but several

potential isoforms can be generated through alternative splicing.

E-proteins bind DNA as either homo- or heterodimers with other members of the HLH

family (Murre, 1992). In nonlymphoid developmental systems the E-proteins form

heterodimers with class II bHLH proteins. In contrast, E-proteins function mainly as

homodimers or heterodimers with other E-proteins in lymphocyte development (Bain et al.,

1993; Sawada and Littman, 1993; Shen and Kadesch, 1995; Sloan et al., 1996). The role of

E-proteins in lymphocyte development is discussed later.

Class II bHLH proteins have a tissue specific expression pattern and can form

heterodimers with the E-proteins (Littlewood, 1998). They are important in neurogenesis,

myogenesis and hematopoiesis. The muscle specific bHLH protein MyoD is one of the most

studied models of E-protein regulation (Lassar et al., 1991). Other class II proteins

important in muscle differentiation are Myf5, MFR4 and myogenin (Arnold and Winter,

1998). A group of proteins that has a role in neuronal differentiation includes NeuroD,

MASH, NSCL, MATH and neurogenin (Guillemot, 1999). The SCL/Tal1 protein is

essential for hematopoietic and vascular development in vertebrates (Shivdasani et al.,

1995; Porcher et al., 1996; Visvader et al., 1998).

A third class consists of the negatively acting HLH proteins, of which there are four

vertebrate Id proteins, Id1, Id2, Id3 and Id4 (Id for “inhibitor of DNA-binding”) (Benezra et

al., 1990; Christy et al., 1991; Sun et al., 1991; Riechmann et al., 1994). Members of this

30

group lack the basic region and can therefore not bind to DNA, when they heterodimerize

with other bHLH proteins they consequently form non-functional complexes. The four Id

proteins are broadly expressed and they dimerize efficiently with E-proteins while their

affinity for class II proteins varies (Riechmann et al., 1994; Langlands et al., 1997).

The fourth class is the bHLHZip proteins, which contain an additional zipper motif C-

terminal of the bHLH domain. The myc, max and mad proteins are members of this group

and they are involved in regulation of cell growth and proliferation (Bernards, 1995). The

bHLH-PAS group of proteins is the fifth class and is characterized by a PAS-domain C-

terminal of the bHLH domain (Crews, 1998).

Regulation of E-protein activity

E-protein activity is regulated at various levels. For example, E-proteins are broadly

expressed, however certain E-protein complexes are restricted to specific cell types. E2A

homodimers dominate in B lineage cells, although heterodimers of E47 and E2-2 have been

noted as well (Murre et al., 1991; Bain et al., 1993; Shen and Kadesch, 1995). E47/HEB

heterodimers are found mostly in thymocytes (Sawada and Littman, 1993; Bain et al.,

1997a). E12 and E2-2 are also expressed in developing thymocytes and B cells

(Corneliussen et al., 1991; Bain et al., 1993), additional complexity is generated by the

presence of distinct HEB and E2-2 isoforms that lack N-terminal transactivation domains

(Corneliussen et al., 1991; Rothenberg, 2002).

E-protein DNA binding activities are inhibited by the Id HLH proteins. Id proteins

efficiently heterodimerize with E-proteins and thus act as dominant-negative regulators

(Benezra et al., 1990; Sun et al., 1991; Riechmann et al., 1994; Langlands et al., 1997; Yan

et al., 1997), the ratio of E-proteins to Id proteins will determine the level of E-protein

activity. Of the four Id proteins, Id2 and Id3 are the main regulators of E-protein activity in

lymphocyte development (Pan et al., 1999; Yokota et al., 1999; Kee et al., 2000; Rivera et

al., 2000).

31

The DNA binding of E-proteins is also inhibited by the calcium/calmodulin complex

(Saarikettu et al., 2004). Calmodulin is a ubiquitously expressed regulator of several cellular

processes. Upon lymphocyte receptor signalling, leading to increased intracellular Ca2+

levels, calmodulin is able to bind and modify target proteins (Van Eldik, 1998).

Phosphorylation of E47 has also been suggested to regulate E-protein activity. In B cells,

E47 seems to be dephosphorylated at specific residues, potentially promoting

homodimerization (Sloan et al., 1996).

E-proteins activate or repress gene expression (Ikawa et al., 2004). The AD1 and AD2

domains mediate the transactivation activities (Aronheim et al., 1993; Quong et al., 1993;

Massari et al., 1999). The AD1 domain can also repress transcription by recruitment of a

family of corepressors called ETO (Zhang et al., 2004). ETO gene products can, like the Id

proteins, inhibit the activity of E-proteins, but whereas the Id proteins affect all E-proteins,

ETO members may only be recruited to a subset of genes in order to mediate transcriptional

repression.

E-proteins in B cell development

Cytokine receptors Flk2/Flt3 and IL-7R, and transcription factors PU.1, Ikaros, E2A, EBF

and Pax5 are critical in the development of B cell precursors (Singh et al., 2005).

Specification of the B lymphoid fate starts as a consequence of expression of Flk2/Flt3

(Mackarehtschian et al., 1995; Adolfsson et al., 2001; Sitnicka et al., 2002). The

transcription factors PU.1 and Ikaros are required for expression of this receptor. Flk2/Flt3

signalling in concert with PU.1 then induces IL-7R (Nichogiannopoulou et al., 1999;

DeKoter et al., 2002; Medina et al., 2004). Combined loss of Flk2/Flt3 and IL-7R results in

complete failure to develop B lineage cells (Sitnicka et al., 2003a; Vosshenrich et al., 2003).

PU.1 is essential for generation of B cells but only when expressed at reduced

concentrations, as high concentrations favour macrophages (DeKoter and Singh, 2000).

Signalling through the IL-7R is proposed to induce the E-protein E2A (Singh et al., 2005).

32

E-proteins, EBF and Pax5 are critical in regulating the early B lineage program of gene

expression (Busslinger, 2004). E2A proteins function in common lymphoid progenitors

upstream of EBF and Pax5 (Kee and Murre, 1998), where they are required for the initiation

of expression of EBF and a subset of B-lineage genes. E2A can induce recombination at the

IgH locus and activation of target genes through recruitment of the SAGA histone-

acetyltransferase complex, which is involved in chromatin remodelling and transcriptional

activation (Massari et al., 1999). In a recent report it was shown that EBF is capable of

rescuing E2A-deficient progenitor cells up to the late pro-B cell stage, confirming that EBF

is the main target for E2A (Seet et al., 2004). PU.1 knockout progenitor cells can also be

rescued by ectopic expression of EBF, suggesting that EBF is downstream of PU.1 (Medina

et al., 2004). It remains to be determined whether E2A and PU.1 work independently or

cooperate in driving the expression of EBF, and whether additional lineage-restricted

transcription factors aid in this process. Conditional PU.1 knock-out mice, where PU.1 is

specifically deleted in the B lineage, do not have any effect on B cell development or

function suggesting that PU.1 is not essential beyond the pre-B stage (Polli et al., 2005).

Once EBF is induced, it acts together with E2A to induce expression of Pax5 (Sigvardsson

et al., 1997; Kee and Murre, 1998; O'Riordan and Grosschedl, 1999), which in turn induces

the expression of B-lineage specific genes, including BLNK, CD19, LEF1 and Igα (Nutt et

al., 1998; Nutt et al., 1999; Schebesta et al., 2002; Souabni et al., 2002). EBF, in

cooperation with RUNX1, modulates DNA methylation and chromatin accessibility in order

to facilitate activation of the Mb-1 gene by Pax5 (Maier et al., 2004). In addition to up

regulating Pax5, E2A and EBF regulate expression of the B cell-specific genes λ5, VpreB,

Igα, Igβ, RAG-1 and RAG-2 (Sigvardsson et al., 1997; Kee and Murre, 1998; O'Riordan

and Grosschedl, 1999).

E2A and Pax5 also inhibit the expression of genes that are normally induced in alternative

hematopoietic lineages. E2A proteins inhibit the expression of genes encoding proteins

involved in myeloid and erythroid development (Ikawa et al., 2004). Pax5 acts to suppress

T-lineage-specific and myeloid-specific gene expression, including Notch1 (Nutt et al.,

1999; Schebesta et al., 2002; Souabni et al., 2002). A recent report has shown that one

33

important function of Pax5 is to repress Flt3 transcription in B cell progenitors and that this

repression is essential for normal B lymphopoiesis (Holmes et al., 2006).

Although E2A and EBF are absolutely essential for the formation of pro-B cells, true

commitment to B cell differentiation is only achieved after expression of Pax5, since Pax5

deficient pro-B cells show a remarkable plasticity and can differentiate into several other

lineages (Nutt et al., 1999; Rolink et al., 1999; Schaniel et al., 2002). To preserve B cell

identity, Pax5 activity needs to be continuously maintained (Horcher et al., 2001; Mikkola

et al., 2002).

As described above, B cell development is critically dependent on E-protein activity and

E2A deficient mice have a complete arrest in development at an early stage, before onset of

immunoglobulin heavy-chain rearrangement (Bain et al., 1994; Zhuang et al., 1994). Such

mice lack early B lineage specific transcripts like Igα, Iµ, RAG1 and Pax5 in their bone

marrow cells. In contrast myeloid development is normal. A similar phenotype is seen in

mice transgenic for Id1, an inhibitor of E-protein activity (Sun, 1994). Ectopic expression of

Id3 in human bone marrow cells results in an analogous block, which demonstrates the

conserved function of E-proteins in murine and human lymphopoiesis (Jaleco et al., 1999).

After promoting B cell commitment, E-proteins control the proliferation of pro-B cells

(Herblot et al., 2002). E2A proteins were shown to be required during the IL-7 dependent

expansion and survival of pro-B lymphocytes (Kee and Murre, 2001). A recent report

demonstrated that E2A proteins are required, in part, due to a role in optimal expression of

N-myc (Seet et al., 2004), a transcription factor involved in cell proliferation and a known

target for IL-7R signalling (Morrow et al., 1992). Signalling through the pBCR then leads

to a transient decrease in E2A expression (Schebesta et al., 2002; Quong et al., 2004) and a

burst of proliferation. E47 expression is increased again in the large, proliferating pre-B

cells, in order to cause cell cycle arrest, induce RAG1/2 expression and to promote Igκ light

chain gene accessibility at the small pre-B cell stage (Romanow et al., 2000; Hsu et al.,

2003; Quong et al., 2004). In immature B cells, E47 remains abundant to promote receptor

editing (Quong et al., 2004). E47 decreases in transitional B cells and even further in

34

IgM+IgD+ B cells. One model proposes that abundant E47 enforces the B cell checkpoint,

allowing continued receptor revision, while signalling from the BCR results in decreased

amounts of E47 and developmental progression (Quong et al., 2004).

A fraction of older mice have a substantial decrease in E47 in pro-B cells that correlates

with a loss of pro-B cells with age (Frasca et al., 2003; Van der Put et al., 2004). It is not

clear if it is the decrease in E47 that is responsible for the reduction in pro-B cell production

or if it is a secondary effect.

In the periphery, high E2A activity favours the development of follicular B cells rather than

MZ B cell development. The opposite is observed when Id3 expression is induced (Quong

et al., 2004). Notch and the adaptor protein Ras modulate the follicular versus MZ B cell

fate (Guinamard et al., 2000; Tanigaki et al., 2002), but it is not know if E2A and Id3 are

linked to these pathways. Furthermore, it has been demonstrated that E2A plays additional

roles during peripheral B lymphopoiesis as E2A expression is induced upon B cell

activation (Quong et al., 1999). It has been shown that E2A proteins are required for

immunoglobulin class switching and somatic hypermutation by directly regulating the

expression of activation-induced deaminase (AID) (Sayegh et al., 2003). It is suggested that

Id2 functions in activated B cells to inhibit E-protein activity once the class switching and

hypermutation is completed (Gonda et al., 2003).

Generation of E2-2 and HEB knockout mice revealed that these genes are not essential for

the establishment of the B cell lineage, rather that B cell development is dependent on these

proteins as the numbers of pro-B cells are reduced in E2-2 and HEB deficient embryos

(Zhuang et al., 1996).

Several lines of evidence suggest considerable redundancy among the different E-proteins.

For example, mice heterozygous for both E2A and HEB or E2-2 have a notable reduction of

B lymphopoiesis compared to mice only heterozygous for E2A (Zhuang et al., 1996).

Moreover, introducing two copies, but not a single copy, of the HEB gene could partially

rescue B cell development in E2A deficient mice (Zhuang et al., 1998). These observations

35

form the basis of the idea that the overall timing and dosage of E-protein activity modulates

a given E-protein regulated event. This would in turn be determined by the amount of

available E-protein and the E/Id protein ratio.

The E-protein knockout mice die at an early age for unknown reasons (Bain et al., 1994;

Zhuang et al., 1994; Zhuang et al., 1996). Only a few E2A and HEB deficient mice survive

to adulthood while all E2-2 deficient mice die around birth. This is unlikely to be a result of

defects in lymphocyte development, but reflects other functions for the E-proteins during

embryonic or postnatal development.

E-proteins in T cell development

The development of T cells is, like B cell development, dependent on E-proteins. However,

unlike B cell development there is no absolute requirement for any of these proteins as T

cells are still generated, albeit at reduced levels, in E-protein deficient mice. Enforced

expression of Id3 in human T lineage precursors and expression of a dominant negative

form of HEB both suppress thymocyte development (Heemskerk et al., 1997; Barndt et al.,

2000), indicating the importance of E-proteins in T cell development.

E47 expression is low at the DN1 stage but is induced during the transition to the DN2 stage

(Engel et al., 2001). The partial block in E2A deficient mice occurs, in αβ T cells, at the

early DN1 stage, before any rearrangement of the β-chain (Bain et al., 1997a). The

transition of thymocytes from the DN1 stage to the DN2 and DN3 subsets is associated with

onset of TCR rearrangement and restriction to the T lineage (Godfrey et al., 1994; Petrie et

al., 1995; Wu et al., 1996). E2A proteins are also important for the generation of γδ T cells,

in part by regulating V(D)J recombination (Bain et al., 1999a). These data indicate that E2A

is necessary for efficient initiation of the T cell developmental program.

In the DN2 and DN3 compartments, E-proteins have multiple functions. They act to initiate

and/or maintain T-lineage specific gene transcription (Schlissel et al., 1991; Herblot et al.,

2000). It has also been reported that E2A directs early events such as V(D)J recombination

in T cell development (Kim et al., 1999; Barndt et al., 2000). In addition, E47 expression is

36

required to suppress developmental progression to the DP stage in the absence of pTCR

signalling (Engel et al., 2001). E47 functions to block cell cycle progression in DN3 cells,

before the expression of TCRβ (Engel and Murre, 2004). Id3 gene expression is induced by

pTCR signalling and E47 levels decrease once cells reach the DP stage (Engel et al., 2001).

Thus, successful β-selection requires down regulation of E47 activity by signalling from the

pTCR complex.

At the DP stage, E-proteins and their antagonist Id3 function to regulate positive selection

(Rivera et al., 2000). Positive selection and maturation of thymocytes at the DP stage is

enhanced in E47 deficient mice (Bain et al., 1999a). Id3 has a reversed phenotype, with

repressed positive selection and maturation (Rivera et al., 2000).

HEB and E2-2 are involved in the regulation of T cell development as well, however T cells

are still generated in HEB and E2-2 deficient mice (Zhuang et al., 1996). In HEB knockout

mice T cell development is affected at the DN to DP stage with an accumulation of ISP cells

(Zhuang et al., 1996; Barndt et al., 1999). It is possible that HEB is necessary for the

transition of thymocytes into the DP stage, alternatively, HEB might be required for

induction of CD4 expression in DP cells (Sawada and Littman, 1993). HEB is also

necessary for the expression of the pTα gene as demonstrated in HEB deficient and

SCL/LMO transgenic mice (Herblot et al., 2000). E2-2 has a reported role at or before the

DN stage in T cell development (Bergqvist et al., 2000).

E2A proteins have been implicated in cellular proliferation and apoptosis. Studies have

shown that both high and low levels of E2A activity promote cell death suggesting that

lymphocytes can only survive within a limited range of E2A activity (Engel and Murre,

1999; Kim et al., 1999; Park et al., 1999). E2A deficient mice develop, in addition to T cell

defects, T cell lymphomas after three to nine months of age (Bain et al., 1997a). The

lymphomas are CD4CD8 DP, of clonal origin, have increased levels of c-myc transcripts

and show chromosomal abnormalities. The mechanism for tumour development is not

known, but when E2A is ectopically expressed in cell-lines derived from E2A deficient

lymphomas, cell death results (Engel and Murre, 1999). Likewise, ectopic expression of

37

E47 in human T cell acute lymphoblastic leukaemia (T-ALL) results in a block in cell cycle

progression and activated programmed cell death (Park et al., 1999). These observations

suggest that E2A might function as a tumour suppressor and that its inactivation is a key

factor in the development of human T-ALL.

Signalling pathways regulating E-protein function

Data from signalling studies suggest that E-proteins control the DP checkpoint in T cell

development. TCR-mediated signalling inhibits the DNA-binding activity of E2A-HEB

heterodimers, in part by the induction of Id3 expression (Bain et al., 2001). Crosslinking of

the TCR leads to activation of the extracellular signal-regulated kinase (Erk) signalling

pathway. Erk activates the transcriptional regulator SAP-1, which in turn induces expression

of Egr-1, that can directly activate Id3 expression (Bain et al., 2001; Bettini et al., 2002;

Costello et al., 2004). Similarly, signalling mediated by transforming growth factor-β (TGF-

β) and bone morphogenetic protein (BMP) activates Id3 expression by activation of

receptor-regulated SMAD transcription factors (Kee et al., 2001).

Notch signalling has been linked to both T cell commitment and MZ versus follicular B cell

development (Pui et al., 1999; Radtke et al., 1999; Tanigaki et al., 2002). The Notch

pathway promotes T cell development and suppresses B cell development by inhibiting E2A

activity via several mechanisms (Ordentlich et al., 1998; Maillard et al., 2003). It was

initially shown that Notch has the ability to modulate the transactivation activity of E47

(Ordentlich et al., 1998). Later studies have shown that Notch signalling promotes E47

degradation after modification by Erk MAP kinase activities (Nie et al., 2003) and that the

carboxyl terminus of Hsc70-interacting protein (CHIP) directly binds E2A and is a crucial

factor in Notch-induced ubiquitination and degradation of E2A proteins (Huang et al.,

2004).

38

Aims of the study

The overall aim of this thesis has been to investigate the genetic control of the mouse

lymphocyte development. The specific questions were:

∗ What is the role of Dµ in B cell development?

∗ How is E2-2 involved in B- and T lymphocyte development?

Results and discussion

Paper I: The role of Dµ protein in B cell development

Before IgH gene rearrangement is completed, a product translated from the DHJH gene

rearranged in reading frame 2 (RF2) (Ichihara et al., 1989), the Dµ protein, can be expressed

in pro-B cells (Reth and Alt, 1984). It has been suggested that this Dµ protein, in association

with the SLC, has some signalling properties similar to the pBCR, such as suppression of

VH to DHJH gene rearrangement and up regulation of light chain gene rearrangement and

expression (Reth et al., 1985; Gu et al., 1991; Tsubata et al., 1992; Ehlich et al., 1994;

Loffert et al., 1996; Tornberg et al., 1998). There is an under representation of

rearrangements in RF2 in mouse pre-B or B cells that is referred to as Dµ selection. The

selection against Dµ is dependent on an intact Dµ transmembrane domain (Gu et al., 1991),

λ5 (Kitamura et al., 1992), Igβ (Gong et al., 1996), the Syk tyrosine kinase (Cheng et al.,

1995) and BLNK/BASH (Hayashi et al., 2003). This suggests that the counter-selection

against Dµ expressing cells is mediated by the deposition of the protein at the cell surface

and that it is dependent on a signalling competent Dµ-pBCR complex. The selection against

RF2 occurs in B220+ CD43+ precursors, before any light chain rearrangements have taken

place, this goes against the possibility that Dµ is unable to pair with a mature light chain,

instead it argues that a pBCR containing Dµ is defective in mediating developmental

39

progression of pro-B cells (Gu et al., 1991; Ehlich et al., 1994; Gong et al., 1996; Horne et

al., 1996). This idea is supported by the analysis of Dµ transgenic mice (Tornberg et al.,

1998; Malynn et al., 2002).

In this study, we wanted to determine the precise developmental stage where expression of

Dµ would block B lymphocyte development. This was performed by breeding Dµ

transgenic mice with mice deficient for RAG2, so that no further rearrangements could

occur. In addition, we wanted to examine if the defect in mediating developmental

progression, could stem from a failure of Dµ to mediate a survival signal to the pro-B cells.

This was done by breeding the Dµ+Rag2-/- mice with mice in which the B cells were

expressing the anti-apoptotic factor Bcl-2 as a transgene. We observed that the Dµ

expressing B cells blocked at the pro-B cell stage (B220+CD43+), more precisely stage C,

assessed by cell surface markers c-Kit, CD2 and CD25. This is in contrast to the study by

Malynn and colleagues who found that the B cells accumulate in stage C´ when expressing

Dµ protein in a rearrangement deficient background (Malynn et al., 2002). However, in

neither of the studies could Dµ mediate progression to the B220+CD43- pre-B cell stage. It

is possible that different Dµ-constructs differ in their ability to block pro- to pre-B cell

transition, either by varying in expression levels or their abilities to associate with SLCs.

This would in turn affect the membrane targeting of the Dµ-pBCR and possibly its function.

When we introduced Bcl-2 into Dµ+Rag2-/- mice, there was an increase in survival and some

accumulation of CD43- cells. These cells are, however, not true pre-B cells, but rather cells

arrested at the pro-B cell stage based on their surface expression of c-Kit, CD2 and CD25.

These cells have previously been referred to as CD43dull cells, and they arise when Bcl-2 is

over expressed in Rag-/- mice (Young et al., 1997). Thus, the Dµ protein does not allow

progression of B cell development beyond that in Rag2-/- mice despite enforced survival by

Bcl-2, and the difference in signalling between µ and Dµ is more fundamental than an

impaired survival signal.

40

The signal transduction pathways from the pBCR and Dµ complexes have not been studied

intensively, in part due to the fact that only a few B cell precursors are expressing such

receptors. Analyses of pre-B cell lines have revealed that pBCR crosslinking induces

tyrosine phosphorylation, lipid raft association, and activation of signalling molecules

involved in BCR signalling, these include the signalling components Igα and Igβ, as well as

the tyrosine kinase Syk (Cheng et al., 1995), the Src kinase family kinases, Fyn, Lyn and

Blk (Saijo et al., 2003) and the linker molecule BLNK, also known as BASH (Pappu et al.,

1999).

There are also several important differences between pBCR and BCR signalling. Firstly, in

pre-B cells there are functional homologs of the signalling molecules Syk and BLNK, that

are normally expressed exclusively in T cells, Zap-70 (Schweighoffer et al., 2003) and

SLP76/LAT (Su and Jumaa, 2003). Secondly, while the Igα ITAM tyrosines of the BCR are

indispensable for peripheral BCR function, they are largely dispensable for early B cell

development (Kraus et al., 1999; Kraus et al., 2001), suggesting that signals may be initiated

differently. Thirdly, the pBCR is not antigen specific and cannot respond to ligands to the

same extent as the peripheral BCR.

It can be speculated that the Dµ-pBCR, because of its truncated VH chain, cannot respond to

a ligand in the same way as the µ-pBCR. Stromal cells have been described as ligands for

pBCR signalling, the human pBCR can interact with galectin (Gauthier et al., 2002), while

the murine pBCR binds heparan sulfate (Bradl et al., 2003). The functional significance of

these interactions is unclear. It is also possible that the pBCR self-aggregates on the cell-

surface. However, most studies indicate that a selecting ligand for the pBCR is not required

for B cell development, but simple surface expression of the pBCR may be sufficient

(Bannish et al., 2001). Studies even suggest that the extracellular domains of the pBCR are

largely irrelevant for mediating B cell development (Shaffer and Schlissel, 1997; Muljo and

Schlissel, 2002). Even if surface expression of pBCR is enough for early B cell

development, it is likely that some aggregation of surface receptor complexes occurs. In

human pre-B cell lines, approximately 30% of resting pBCR accumulate in lipid rafts,

whereas in mature B cell lines the BCR is excluded from lipid rafts (Guo et al., 2000). This

41

would serve to concentrate receptor complexes and bring them into proximity with other

signalling molecules. Together, these studies makes it unlikely that the difference in

signalling between Dµ and µ is mediated by the ability to bind a ligand, but more likely the

ability to aggregate in lipid rafts is affected.

Another alternative is that µ-pBCR and Dµ-pBCR might generate qualitatively different

signals. Recent analyses of mice that are deficient in BLNK/BASH indicate that the pBCR

activates two or more signalling pathways, each with a different functions in B cell

development. The study by Hayashi and colleagues has demonstrated that while neither

CD19 nor BLNK is necessary for IgH allelic exclusion, BLNK is essential for Dµ selection,

thus distinct signals are required for these events (Hayashi et al., 2003). Since Syk has been

shown to be necessary for Dµ selection (Cheng et al., 1995), it is likely that Syk-

phosphorylated BLNK recruits signalling molecules mediating Dµ selection. Since CD19

does not appear to complement BLNK in Dµ selection, the Dµ complex signalling

mechanism appears to also be distinct from the pBCR signalling mechanism promoting pre-

B cell transition.

An alternative to different signalling pathways, one for differentiation and one for allelic

exclusion and light chain rearrangements, is the possibility that the quantity of one signal

determines cell fate. Maybe Dµ is less efficient than µ in inducing a signal because of its

different conformation.

Interestingly, when Dµ is expressed as a transgene on a recombination competent

background, the block in B cell development allows MZ B cell development whereas the

formation of follicular B cells is severely affected. The cells escaping the Dµ-induced block

seem to require an endogenous functional V(D)J rearrangement since they express IgM on

their cell surface and since no cells can pass the pro-B cell stage in Dµ+Rag2-/- mice. It is

possible that the low surface expression of Dµ that we can detect on primary B cells would

allow for continued rearrangement, alternatively the rearrangements were done before Dµ

was fully expressed on the cell surface. Incomplete allelic exclusion can also be observed in

42

µ-transgenic mice, where it is confined to the most D-proximal VH-genes, such as 7183

(Costa et al., 1992). It is possible that B cells in Dµ transgenic mice are rescued by a

rearrangement in the D-proximal genes and that this promotes the relative increase in MZ B

cell formation.

Increased MZ formation is also observed when the B cell output from the BM is inhibited

by induced Rag deletion (Hao and Rajewsky, 2001), as well as in other models where B cell

development is compromised (Carvalho et al., 2001; Quong et al., 2004). The proportionally

large MZ in Dµ transgenic mice might therefore reflect homeostatic mechanisms that

function to give priority to the effector branch of the B cells.

Paper II: The role of E2-2 in B cell development

As discussed previously, differentiation, survival and population expansion of B and T

lineage cells in the bone marrow, thymus and peripheral lymphoid organs are regulated at

least in part by bHLH proteins. In B lymphocyte development, the E-proteins have several

important roles. Targeting experiments have shown that E2A deficient mice have severe

effects on B cell development, while HEB and E2-2 deficient mice have only mild effects

(Bain et al., 1994; Zhuang et al., 1994; Zhuang et al., 1996). Compared to E2A and HEB,

the role of E2-2 in B cell development is poorly characterised.

In paper II, we set out to analyse the expression pattern of E2-2, in addition to E2A and

HEB, in order to determine the role of E2-2 in B cell development in more detail. Different

B cell populations were sorted from wild type mice and analysed by Real time quantitative

PCR (TaqMan). E2-2 was predominantly expressed at the pro-B cell stage, indicating a

function for E2-2 at this early stage. E2A was also expressed at the pro-B cell stage, but

even though E2-2 expression levels seemed higher than E2A at this stage, it is difficult to be

certain due to the use of different TaqMan assays. E2A has been shown by others to have a

role in early B cell development, the development in E2A-/- mice is blocked before any

rearrangements have taken place (Bain et al., 1994; Zhuang et al., 1994). Besides the

function in commitment to the B cell lineage, E2A is also required during the IL-7

43

dependent expansion and survival of pro-B lymphocytes (Kee and Murre, 2001), in part due

to E2A’s role in optimal expression of N-myc (Seet et al., 2004), a transcription factor

involved in cell proliferation and a known target for IL-7R signalling (Morrow et al., 1992).

We estimated the frequency of IL-7 responding cells by growing E2-2-/- FL cells on S-17

stromal cells supplemented with IL-7. E2-2 deficiency renders a reduced frequency of

responding B cell progenitors, despite normal IL-7Rα surface expression.

E2A expression increased in the transition between the pro-B and the pre-B cell stages in

our study. E2A is known to induce RAG1/2 expression and to promote Igκ light chain gene

accessibility at the small pre-B cell stage (Romanow et al., 2000; Hsu et al., 2003; Quong et

al., 2004). In immature B cells, E47 remains abundant to promote receptor editing (Quong

et al., 2004) but as we observed, E2A expression decreases in mature B cells in the spleen.

HEB expression was low at all stages, indicating a minor role in B cell development.

However, the number of pro-B cells was reduced in HEB deficient embryos (Zhuang et al.,

1996) and a tandem copy of HEB can partially compensate for E2A deficiency (Zhuang et

al., 1998). This implies that the expression pattern is a principal functional determinant for

the E-proteins and supports a threshold model for E-protein activity. A minimum E-protein

threshold must be exceeded for B lineage entry and efficient pro-B cell expansion, but cells

are further selected on the basis of total E-protein expression. However, despite high levels

of expression at the pro-B cell stage, E2-2 cannot compensate for a defect in E2A, hence

expression levels cannot explain all functional differences.

Mice deficient in E2-2 die at an early age, making studies of adult mice impossible. In order

to determine where E2-2 functions in B cell development, we therefore analysed the ability

of E2-2 deficient cells to reconstitute an empty host by injecting fetal liver (FL) cells into

irradiated Rag2-/-γc-/- mice. In this model, E2-2 deficiency leads to an impaired B cell

expansion. The numbers of pro- and pre-B cells in the BM decreased by 40-50% and

peripheral IgM+ cells decreased by 30%. This indicates that defects stemming from E2-2

deficiency manifest at an early stage and are then slightly compensated for in the peripheral

B cell compartments. Interestingly, there was no difference in the number of IgM+ cells

from mice reconstituted with E2-2+/- versus E2-2-/- FL cells. This could be interpreted as the

44

E2-2-/- B cells have a slight advantage in the periphery. Declining levels of E47 at the

immature B cell stage has been suggested to promote developmental progression (Quong et

al., 2004). Thus, it is possible that the small reduction in E-protein activity at the immature

B cell stage of E2-2-/- mice results in a slight advantage of B cells to leave the BM. It could

also reflect compensatory homeostatic mechanisms, promoting formation of MZ B cells, as

observed when the B cell output from the BM is inhibited by induced Rag deletion (Hao and

Rajewsky, 2001).

When the peripheral B cell compartments were analysed in reconstituted mice, we noticed

that the number of MZ B cells were unaffected by a defect in E2-2, while the follicular B

cell development was severely perturbed, resulting in a relative increase in MZ B cells. This

has previously been observed in E2A+/- mice and it has been suggested that E2A plays a role

in the MZ versus follicular decision (Quong et al., 2004). Upon analysing the expression of

E2-2 and E2A in MZ and follicular B cell populations, we observed a significant increase in

E2-2 expression in follicular B cells. We also saw a tendency towards higher E2A

expression in follicular B cells, but this difference was not significant. It seems likely that

under normal conditions, the follicular versus MZ decision is specifically regulated by E2-2.

The E-protein antagonist Id3 has been suggested to be involved in the MZ/follicular

decision, and a decreased proportion of MZ B cells is observed in Id3 deficient mice

(Quong et al., 2004). In our study, Id3 expression was higher in MZ compared to follicular

B cells. Thus, it might be the concomitant down regulation of E2-2 and up regulation of Id3

that trigger the decision to become a MZ B cell.

The models of MZ B cell development are controversial and the so-called transitional stages

through which MZ B cells develop have been classified in different ways (Loder et al.,

1999; Allman et al., 2001). A recent report by Srivastava et al. challenged these models,

suggesting that MZ B cells develop by way of non-dividing CD21/CD35highCD23+

intermediates (Srivastava et al., 2005). Their model for peripheral B cell maturation

involves two subsets in the spleen, the first being the AA4+ transitional 1 (T1) subset, which

then gives rise to the AA4+ T2 subset. The T2 subset represents the common precursor of

both follicular and MZ B cells. CD21/CD35highCD23+ cells are the immediate precursors of

45

MZ B cells and can arise from T2 cells, follicular B cells, or both. It is possible that the

CD21/CD35highCD23+ subset is in dynamic equilibrium with the follicular subset

(Srivastava et al., 2005). In our study, we have defined follicular and MZ B cells by surface

expression of CD21 and CD23, meaning that we have included the CD21/CD35highCD23+

intermediate cells in the MZ B cell population.

Collectively, our findings point to a role for E2-2 operating at the pro-B cell stage, where

high mRNA expression was observed, to facilitate B cell commitment and to expand early

B lineage cells. In addition, we provided evidence that E2-2 deficient cells regulate the

follicular versus MZ decision, and that this correlates with the modulation of E2-2 mRNA

levels.

Paper III: The role of E2-2 in T cell development

Even though we did not detect any abnormalities in T cell development in the reconstituted

mice discussed in the previous section, data previously generated in the lab indicated that

E2-2 also has a function in T cell development. In mice that are mosaic with respect to E2-2

expression, cells with a homozygous null mutated E2-2 gene are underrepresented in B

lymphocyte as well as T lymphocyte cell lineages (Bergqvist et al., 2000). Such skewing in

mosaicism is already observed in DP thymocytes, indicating a partial block in early T cell

development, when E2-2 is absent (Bergqvist et al., 2000).

In paper III, we analysed the expression of the different E-proteins and their antagonists, Id-

protein 1-4, in different T cell developmental stages. Different T cell populations were

sorted from wild type mice and analysed by Real time quantitative PCR (TaqMan). The

distinct functional effects of the different E-proteins appear to be reflected in their

expression patterns during T cell development. The up regulation of E2A mRNA in DN2

correlates with reported E47 protein levels and its role in the regulation of β-selection

(Engel et al., 2001). Similarly the increased expression of HEB transcript in the DN2, DN3

and DP stages fits well with observed phenotypes at the DN3 stage and in the DN to DP

transition (Barndt et al., 1999; Herblot et al., 2000). High levels of E2-2 RNA were

46

observed at the DN1 and DN2 stages, as well as an up regulation at the DP stage. The fact

that all E-protein transcripts, together with Id3, show some level of up regulation at the DP

stage highlights the complex interplay of this family of transcription factors in the

regulation of negative and positive selection

Since the reconstitution system used in paper II, might be too insensitive for detecting small

T cell abnormalities, we used the fetal thymus organ culture (FTOC) system to study E2-2´s

function in T cell development. This culture system enables T cells to go through

development and accumulate in their thymic environment in vitro, as well as enhances a

possible phenotype upon deletion of E2-2. To verify the system first, we decided to repeat

previous data regarding E2A and HEB. In our study, E2A deficient thymocytes were

partially blocked at the DN1 stage, in addition, there was an accelerated positive selection

with reduced numbers of DP cells and increased numbers of SP cells. This is in line with

previous observations in E2A deficient mice (Bain et al., 1997a; Bain et al., 1999a). HEB

deficient mice have been shown to accumulate immature single positive cells (ISP) in the

thymus (Zhuang et al., 1996; Barndt et al., 1999), which was also observed in our

experimental system. In addition we saw an accumulation of cells at the DN3 stage. HEB

has previously been shown to be involved in the regulation of pTα at this stage (Herblot et

al., 2000; Reizis and Leder, 2001; Takeuchi et al., 2001; Petersson et al., 2002; Tremblay et

al., 2003).

When applying the FTOC system to E2-2 deficient cells, we observed an accumulation at

the DN3 stage, indicating a partial block there. Thus, E2-2 deletion leads to an α/β

thymocyte developmental defect in line with those elicited by other E-proteins in our

experimental system. We observed no affect of E2-2 on γδT cell development, in

accordance with similar observations for HEB, however E2A deficient mice have been

shown to have a severe impairment in γδT cell development (Bain et al., 1999b; Barndt et

al., 1999).

The fact that there are no differences in the other stages, except for in DN3, when E2-2 is

missing during FTOC is puzzling, but might reflect the fact that E2-2 deletion only results

47

in a fragile phenotype. Actually, the block at the DN3 stage in E2-2-/- mice could not be seen

ex vivo, but only after FTOC. This might explain why we could not detect it in the mice

reconstituted with E2-2 deficient FL cells. Another possible explanation as to why we might

not detect any block in thymocyte development in these mice is that the defect is not cell

autonomous but rather due to thymic stroma being E2-2-/-. This could be resolved by doing

reconstitution thymus organ culture (RTOC), which should have the same sensitivity as the

FTOC to detect any abnormalities in T cell development.

Upon analysing the E2-2-/- thymocytes, we saw that the DN3 blocked thymocytes express

TCRβ but have reduced levels of pTα. The pTα enhancer is known to be regulated by a

complex combination of transcription factors including c-Myb, CSL-NICD complex (Reizis

and Leder, 2001; Reizis and Leder, 2002) and E-proteins E2A and HEB (Herblot et al.,

2000; Reizis and Leder, 2001; Takeuchi et al., 2001; Petersson et al., 2002; Tremblay et al.,

2003). Relative levels of E2A, HEB and their inhibitor SCL have been reported to control

pTα gene expression in immature thymocytes (Tremblay et al., 2003) and over expression

of HEB and E2A in thymocyte cell lines activates pTα activity (Takeuchi et al., 2001). Our

data suggests that E2-2 could be included in this network of factors regulating pTα levels.

E2A/HEB heterodimers have been shown to predominate in the thymus (Sawada and

Littman, 1993; Bain et al., 1997b), however, it is possible that E2-2 can heterodimerise with

E2A and/or HEB, and in this way add to the regulation of pTα expression. Even though E2-

2 expression is turned on before β-selection, the accumulation of cells at the DN3 stage in

E2-2-/- mice is reasonable given that E2-2´s effect is mediated through the control of pTα

expression, which in turn is required for the cells to proceed through β-selection and enter

the DN4 stage.

The thymocytes blocked at the DN3 stage proliferate and go through apoptosis at normal

levels and are not causing the accumulation of cells. Normally, cells that can not pass β-

selection die through apoptosis, but the fact that we did not detect an increase in apoptosis at

the β-selection stage might be because E2-2 functions as a regulator of β-selection in a

48

similar way as reported for E2A (Engel et al., 2001) so that in the absence of E2-2,

thymocytes with defects in pTCR are allowed to develop.

In conclusion, E2-2 functions in the balance between E-proteins and their inhibitors for

correct licensing of developing T lymphocytes, both in β-selection of immature thymocytes

and in negative/positive selection of α/β T cells.

Many transcription factors controlling various stages of B- and T cell development and

function have been identified, but there are still other important factors to discover. It has

also become clear that transcription factors can work together to form regulatory networks.

An important area that requires a lot of future work is to identify the crucial target genes for

many of the transcription factors.

49

Conclusions • The Dµ protein does not allow progression of B cell development beyond that in Rag2-/-

mice, despite enforced survival with Bcl-2

• In Dµ-transgenic mice, the Dµ induced partial block allows MZ B cell development

whereas the formation of follicular B cells is severely reduced

• E2-2 deficiency renders a reduced frequency of IL-7 responsive B cell progenitors, despite

normal surface expression of IL-7Rα

• A relative increase in MZ B cells in the periphery of E2-2-/- reconstituted mice correlates

with the differential expression pattern of E2-2 in follicular versus MZ B cells

• The difference in expression between various E-proteins corresponds to their functions

during B and T cell development

• E2-2 deletion leads to an α/β thymocyte developmental defect in line with those elicited

by other E-proteins in our experimental system

• Accumulated DN3 thymocytes in E2-2 deficient mice display normal ratios of TCRβ

expression but altered levels of pTα expression

50

Acknowledgements

Firstly, a big thanks to my supervisor Dan Holmberg, who let me start in his group and then helped me through it all with his never ending optimism and good humor. Also a huge thanks to my transient “postdoc-supervisors”: Ulla-Carin, my first mentor, I have not forgotten my rum-raisin chocolate partner, and I don´t think anyone will forget the 81X overhead! Ingela B, we were quite a team, I will always remember when we acquired all of our samples on the FACS and didn’t save the data, we rescued it though…..Mario (in memory), your Latin lab-style could be frustrating at times but never boring! Thank you for also being a good friend. Johan F, thanks for helping with all the papers, by the way, sure you are not part Latin? The people in the group: Sofia, it is great sharing an office with you, no one can chitchat and still be productive in the same way as you (you are efficient enough for two!). Maria E, thank you for sharing the lymphocyte development group with me, and for putting up with all of my questions, I am so impressed by your knowledge. Marie, you are a better scientist than you think (I see some Vini-tendencies in you), I know that you will finish in no time! Thanks for the Friday massages, they are heaven, I will miss them! Vinicius, you are such a dedicated and knowledge-thirsty person and I think you make an excellent scientist, and friend! Nadia, I wish you all the sun and fun that you have missed out on while being in Umeå! Anna LB, keep hanging in there, dissertation is in reach. Good luck in your new house! Beta, you are always willing to help with computer- and dissertation questions, thanks also for the continuous supply of candy from Iceland!! Ann-Sofie, you know the feeling of struggling with a project, persistence is the key;). Sofie, your laugh lights up the lab! Petter, thanks for being a good neighbor, watering our plants while we travel the globe, and for showing me frisbee-golf! Mikael, you have saved my day many times by providing help with computer problems, they always arise mysteriously! Åsa L, you are a star on sorting and injecting mice, thanks for all the help! Anna-Karin, you are an asset to the lab, and such a nice person, thanks for the orienteering lesson! Maria L, always nice to talk to, good luck with your next loppis! Tomas, Susanne, Pia, Lotta, Malin, Ulrika, Pasi, Stefan, Urban, Lisbeth L, Lisbet Ä, Åsa E, thank you all very much for making the fikas such a nice and enjoyable time! Linda, I look forward to some more mountain-walking this summer, Martin, thanks for introducing me to poker, even if it didn’t really stick… Monica, thanks for the brief Friday meetings and for approving the holidays! Anna N, thanks for sharing the agony of organizing a dissertation, I had a lot of help from you! Elin and Regina, keep up the good work! Birgitta, and P-A, thanks for taking care of everything outside the lab, it just wouldn’t work without you two! Terry and Åsa Lundsten, thank you for organizing nice parties and retreats for the department! Thanks also to all the animal caretakers and the Dish and media personnel. Thank you, “Kioskgubben” for the candy supply within convenient reach! Kristina Lejon, thanks for stepping in for Dan, reading my applications and looking through presentations! Mattias Haury and Francesco Colucci, thank you for letting me come and spend some time in your labs! Tomas Grundström and Jannek, thanks for sharing the interest for E-proteins! Dan Hultmark, thank you for taking the time to go through my work and scrutinize it! Elisabeth Pålsson, thanks for struggling with the ES cells at 30 ºC with me!

51

The “beer corner guys”, Vinicius, Mario (in memory), Antony, Jason, Joan, and Dinis thanks for those great Friday evenings. My dear friends: Marie, you are actually my longest friend, thanks for all the listening, talking and understanding during these years, I know that I can always count on you, hope you know the same! Mia S, you are really a “frisk fläkt”, so open and friendly it’s contaminating, you make me a better person! Sofia, thanks for the fikas and deep-talks, seeing you with your children makes me want to have some one day… Vinicius and Tania, my Brazilian friends! I miss the dinners, and the fikas in town. You are both wonderful people and I am happy that our paths have crossed, and I hope they will keep crossing! Lotta and Lisandro, thank you for all the nice parties, dinners (and Lotta, all the soap-evenings;), for showing us the highlights of Piteå and for freezing together at the market in Jokkmokk! Joan, you are lots of fun, craziness and activity, I am happy to also know your other sides and I hope we will always stay friends, ready for an update-fika? Big thanks to Kurt´s family in Australia who made me feel at home when over there, I look forward to seeing you all again! Anneli, thanks for making the time growing up so much more fun, you have been a fantastic little sister! It is great to do fikas, dinners, parties, skiing/snowboarding and renovating with you and Daniel! Mum and dad, thanks for making me believe that I had academic skills when I was young and for letting me borrow your car now when I’m older, thanks also for the nice home-cooked dinners! Last, but not least, I want to thank Kurt, for being supportive, lovable, annoying, funny, charming, critical, encouraging, loving, helpful and so much more during the years together, but most of all, thanks for being the love of my life! You know I would go anywhere with you, lucky for me it’s Australia...!

52

References Abramson, S., R. G. Miller and R. A. Phillips (1977). "The identification in adult bone marrow of pluripotent and restricted stem cells of the myeloid and lymphoid systems." J Exp Med 145(6): 1567-79.

Adolfsson, J., O. J. Borge, D. Bryder, K. Theilgaard-Monch, I. Astrand-Grundstrom, E. Sitnicka, Y. Sasaki and S. E. Jacobsen (2001). "Upregulation of Flt3 expression within the bone marrow Lin(-)Sca1(+)c-kit(+) stem cell compartment is accompanied by loss of self-renewal capacity." Immunity 15(4): 659-69.

Allen, R. D., 3rd, T. P. Bender and G. Siu (1999). "c-Myb is essential for early T cell development." Genes & development 13(9): 1073-8.

Allman, D., R. C. Lindsley, W. DeMuth, K. Rudd, S. A. Shinton and R. R. Hardy (2001). "Resolution of three nonproliferative immature splenic B cell subsets reveals multiple selection points during peripheral B cell maturation." Journal of immunology (Baltimore, Md. 167(12): 6834-40.

Allman, D., A. Sambandam, S. Kim, J. P. Miller, A. Pagan, D. Well, A. Meraz and A. Bhandoola (2003). "Thymopoiesis independent of common lymphoid progenitors." Nature immunology 4(2): 168-74.

Alt, F. W. and D. Baltimore (1982). "Joining of immunoglobulin heavy chain gene segments: implications from a chromosome with evidence of three D-JH fusions." Proc Natl Acad Sci U S A 79(13): 4118-22.

Amit, A. G., R. A. Mariuzza, S. E. Phillips and R. J. Poljak (1985). "Three-dimensional structure of an antigen-antibody complex at 6 A resolution." Nature 313(5998): 156-8.

Amit, A. G., R. A. Mariuzza, S. E. Phillips and R. J. Poljak (1986). "Three-dimensional structure of an antigen-antibody complex at 2.8 A resolution." Science 233(4765): 747-53.

Ardavin, C., L. Wu, C. L. Li and K. Shortman (1993). "Thymic dendritic cells and T cells develop simultaneously in the thymus from a common precursor population." Nature 362(6422): 761-3.

Arnold, H. H. and B. Winter (1998). "Muscle differentiation: more complexity to the network of myogenic regulators." Current opinion in genetics & development 8(5): 539-44.

Aronheim, A., R. Shiran, A. Rosen and M. D. Walker (1993). "The E2A gene product contains two separable and functionally distinct transcription activation domains." Proceedings of the National Academy of Sciences of the United States of America 90(17): 8063-7.

Bain, G., C. B. Cravatt, C. Loomans, J. Alberola-Ila, S. M. Hedrick and C. Murre (2001). "Regulation of the helix-loop-helix proteins, E2A and Id3, by the Ras-ERK MAPK cascade." Nat Immunol 2(2): 165-71.

Bain, G., I. Engel, E. C. Robanus Maandag, H. P. te Riele, J. R. Voland, L. L. Sharp, J. Chun, B. Huey, D. Pinkel and C. Murre (1997a). "E2A deficiency leads to abnormalities in alphabeta T-cell development and to rapid development of T-cell lymphomas." Mol Cell Biol 17(8): 4782-91.

Bain, G., S. Gruenwald and C. Murre (1993). "E2A and E2-2 are subunits of B-cell-specific E2-box DNA-binding proteins." Mol Cell Biol 13(6): 3522-9.

Bain, G., E. C. Maandag, D. J. Izon, D. Amsen, A. M. Kruisbeek, B. C. Weintraub, I. Krop, M. S. Schlissel, A. J. Feeney, M. van Roon and et al. (1994). "E2A proteins are required for proper B cell development and initiation of immunoglobulin gene rearrangements." Cell 79(5): 885-92.

53

Bain, G., M. W. Quong, R. S. Soloff, S. M. Hedrick and C. Murre (1999a). "Thymocyte maturation is regulated by the activity of the helix-loop-helix protein, E47." J Exp Med 190(11): 1605-16.

Bain, G., E. C. Robanus Maandag, H. P. te Riele, A. J. Feeney, A. Sheehy, M. Schlissel, S. A. Shinton, R. R. Hardy and C. Murre (1997b). "Both E12 and E47 allow commitment to the B cell lineage." Immunity 6(2): 145-54.

Bain, G., W. J. Romanow, K. Albers, W. L. Havran and C. Murre (1999b). "Positive and negative regulation of V(D)J recombination by the E2A proteins." The Journal of experimental medicine 189(2): 289-300.

Bank, I., R. A. DePinho, M. B. Brenner, J. Cassimeris, F. W. Alt and L. Chess (1986). "A functional T3 molecule associated with a novel heterodimer on the surface of immature human thymocytes." Nature 322(6075): 179-81.

Bannish, G., E. M. Fuentes-Panana, J. C. Cambier, W. S. Pear and J. G. Monroe (2001). "Ligand-independent signaling functions for the B lymphocyte antigen receptor and their role in positive selection during B lymphopoiesis." The Journal of experimental medicine 194(11): 1583-96.

Barndt, R., M. F. Dai and Y. Zhuang (1999). "A novel role for HEB downstream or parallel to the pre-TCR signaling pathway during alpha beta thymopoiesis." J Immunol 163(6): 3331-43.

Barndt, R. J., M. Dai and Y. Zhuang (2000). "Functions of E2A-HEB heterodimers in T-cell development revealed by a dominant negative mutation of HEB." Mol Cell Biol 20(18): 6677-85.

Benezra, R., R. L. Davis, D. Lockshon, D. L. Turner and H. Weintraub (1990). "The protein Id: a negative regulator of helix-loop-helix DNA binding proteins." Cell 61(1): 49-59.

Bergqvist, I., M. Eriksson, J. Saarikettu, B. Eriksson, B. Corneliussen, T. Grundstrom and D. Holmberg (2000). "The basic helix-loop-helix transcription factor E2-2 is involved in T lymphocyte development." Eur J Immunol 30(10): 2857-63.

Berland, R. and H. H. Wortis (2002). "Origins and functions of B-1 cells with notes on the role of CD5." Annu Rev Immunol 20: 253-300.

Bernard, O., N. Hozumi and S. Tonegawa (1978). "Sequences of mouse immunoglobulin light chain genes before and after somatic changes." Cell 15(4): 1133-44.

Bernards, R. (1995). "Transcriptional regulation. Flipping the Myc switch." Current biology 5(8): 859-61.

Bernasconi, N. L., N. Onai and A. Lanzavecchia (2003). "A role for Toll-like receptors in acquired immunity: up-regulation of TLR9 by BCR triggering in naive B cells and constitutive expression in memory B cells." Blood 101(11): 4500-4.

Berthelot, J. M. and Y. Maugars (2004). "Role for suppressor T cells in the pathogenesis of autoimmune diseases (including rheumatoid arthritis). Facts and hypotheses." Joint Bone Spine 71(5): 374-80.

Bettini, M., H. Xi, J. Milbrandt and G. J. Kersh (2002). "Thymocyte development in early growth response gene 1-deficient mice." Journal of immunology (Baltimore, Md. 169(4): 1713-20.

Bjorkman, P. J., M. A. Saper, B. Samraoui, W. S. Bennett, J. L. Strominger and D. C. Wiley (1987). "The foreign antigen binding site and T cell recognition regions of class I histocompatibility antigens." Nature 329(6139): 512-8.

54

Bosselut, R., L. Feigenbaum, S. O. Sharrow and A. Singer (2001). "Strength of signaling by CD4 and CD8 coreceptor tails determines the number but not the lineage direction of positively selected thymocytes." Immunity 14(4): 483-94.

Bosselut, R., T. I. Guinter, S. O. Sharrow and A. Singer (2003). "Unraveling a revealing paradox: Why major histocompatibility complex I-signaled thymocytes "paradoxically" appear as CD4+8lo transitional cells during positive selection of CD8+ T cells." J Exp Med 197(12): 1709-19.

Bourke, E., D. Bosisio, J. Golay, N. Polentarutti and A. Mantovani (2003). "The toll-like receptor repertoire of human B lymphocytes: inducible and selective expression of TLR9 and TLR10 in normal and transformed cells." Blood 102(3): 956-63.

Bradl, H., J. Wittmann, D. Milius, C. Vettermann and H. M. Jack (2003). "Interaction of murine precursor B cell receptor with stroma cells is controlled by the unique tail of lambda 5 and stroma cell-associated heparan sulfate." Journal of immunology (Baltimore, Md. 171(5): 2338-48.

Brenner, M. B., J. McLean, D. P. Dialynas, J. L. Strominger, J. A. Smith, F. L. Owen, J. G. Seidman, S. Ip, F. Rosen and M. S. Krangel (1986). "Identification of a putative second T-cell receptor." Nature 322(6075): 145-9.

Brodeur, P. H. and R. Riblet (1984). "The immunoglobulin heavy chain variable region (Igh-V) locus in the mouse. I. One hundred Igh-V genes comprise seven families of homologous genes." Eur J Immunol 14(10): 922-30.

Burnet, F. M. (1976). "A modification of Jerne's theory of antibody production using the concept of clonal selection." CA Cancer J Clin 26(2): 119-21.

Busslinger, M. (2004). "Transcriptional control of early B cell development." Annu Rev Immunol 22: 55-79.

Cantrell, D. A. (2002). "T-cell antigen receptor signal transduction." Immunology 105(4): 369-74.

Carlsson, L. and D. Holmberg (1990). "Genetic basis of the neonatal antibody repertoire: germline V-gene expression and limited N-region diversity." Int Immunol 2(7): 639-43.

Carlsson, L., C. Overmo and D. Holmberg (1992). "Developmentally controlled selection of antibody genes: characterization of individual VH7183 genes and evidence for stage-specific somatic diversification." Eur J Immunol 22(1): 71-8.

Carvalho, T. L., T. Mota-Santos, A. Cumano, J. Demengeot and P. Vieira (2001). "Arrested B lymphopoiesis and persistence of activated B cells in adult interleukin 7(-/)- mice." J Exp Med 194(8): 1141-50.

Chaffin, K. E., C. R. Beals, T. M. Wilkie, K. A. Forbush, M. I. Simon and R. M. Perlmutter (1990). "Dissection of thymocyte signaling pathways by in vivo expression of pertussis toxin ADP-ribosyltransferase." Embo J 9(12): 3821-9.

Chang, Y., C. J. Paige and G. E. Wu (1992). "Enumeration and characterization of DJH structures in mouse fetal liver." Embo J 11(5): 1891-9.

Cheng, A. M., B. Rowley, W. Pao, A. Hayday, J. B. Bolen and T. Pawson (1995). "Syk tyrosine kinase required for mouse viability and B-cell development." Nature 378(6554): 303-6.

Christy, B. A., L. K. Sanders, L. F. Lau, N. G. Copeland, N. A. Jenkins and D. Nathans (1991). "An Id-related helix-loop-helix protein encoded by a growth factor-inducible gene." Proc Natl Acad Sci U S A 88(5): 1815-9.

55

Constant, S. L. and K. Bottomly (1997). "Induction of Th1 and Th2 CD4+ T cell responses: the alternative approaches." Annu Rev Immunol 15: 297-322.

Corneliussen, B., A. Thornell, B. Hallberg and T. Grundstrom (1991). "Helix-loop-helix transcriptional activators bind to a sequence in glucocorticoid response elements of retrovirus enhancers." J Virol 65(11): 6084-93.

Costa, T. E., H. Suh and M. C. Nussenzweig (1992). "Chromosomal position of rearranging gene segments influences allelic exclusion in transgenic mice." Proc Natl Acad Sci U S A 89(6): 2205-8.

Costello, P. S., R. H. Nicolas, Y. Watanabe, I. Rosewell and R. Treisman (2004). "Ternary complex factor SAP-1 is required for Erk-mediated thymocyte positive selection." Nature immunology 5(3): 289-98.

Crews, S. T. (1998). "Control of cell lineage-specific development and transcription by bHLH-PAS proteins." Genes & development 12(5): 607-20.

Davidson, H. W., P. A. Reid, A. Lanzavecchia and C. Watts (1991). "Processed antigen binds to newly synthesized MHC class II molecules in antigen-specific B lymphocytes." Cell 67(1): 105-16.

Decker, D. J., N. E. Boyle, J. A. Koziol and N. R. Klinman (1991). "The expression of the Ig H chain repertoire in developing bone marrow B lineage cells." J Immunol 146(1): 350-61.

DeKoter, R. P., H. J. Lee and H. Singh (2002). "PU.1 regulates expression of the interleukin-7 receptor in lymphoid progenitors." Immunity 16(2): 297-309.

DeKoter, R. P. and H. Singh (2000). "Regulation of B lymphocyte and macrophage development by graded expression of PU.1." Science 288(5470): 1439-41.

Doyle, C. and J. L. Strominger (1987). "Interaction between CD4 and class II MHC molecules mediates cell adhesion." Nature 330(6145): 256-9.

Dudley, E. C., H. T. Petrie, L. M. Shah, M. J. Owen and A. C. Hayday (1994). "T cell receptor beta chain gene rearrangement and selection during thymocyte development in adult mice." Immunity 1(2): 83-93.

Dul, J. L., Y. Argon, T. Winkler, E. ten Boekel, F. Melchers and I. L. Martensson (1996). "The murine VpreB1 and VpreB2 genes both encode a protein of the surrogate light chain and are co-expressed during B cell development." Eur J Immunol 26(4): 906-13.

Early, P., H. Huang, M. Davis, K. Calame and L. Hood (1980). "An immunoglobulin heavy chain variable region gene is generated from three segments of DNA: VH, D and JH." Cell 19(4): 981-92.

Edelman, G. M. (1973). "Antibody structure and molecular immunology." Science 180(88): 830-40.

Ehlich, A., V. Martin, W. Muller and K. Rajewsky (1994). "Analysis of the B-cell progenitor compartment at the level of single cells." Curr Biol 4(7): 573-83.

Engel, I., C. Johns, G. Bain, R. R. Rivera and C. Murre (2001). "Early thymocyte development is regulated by modulation of E2A protein activity." J Exp Med 194(6): 733-45.

Engel, I. and C. Murre (1999). "Ectopic expression of E47 or E12 promotes the death of E2A-deficient lymphomas." Proceedings of the National Academy of Sciences of the United States of America 96(3): 996-1001.

Engel, I. and C. Murre (2004). "E2A proteins enforce a proliferation checkpoint in developing thymocytes." The EMBO journal 23(1): 202-11.

56

Ephrussi, A., G. M. Church, S. Tonegawa and W. Gilbert (1985). "B lineage--specific interactions of an immunoglobulin enhancer with cellular factors in vivo." Science 227(4683): 134-40.

Erlandsson, L., S. Licence, F. Gaspal, P. Lane, A. E. Corcoran and I. L. Martensson (2005). "Both the pre-BCR and the IL-7Ralpha are essential for expansion at the pre-BII cell stage in vivo." Eur J Immunol 35(6): 1969-76.

Ernst, P. and S. T. Smale (1995). "Combinatorial regulation of transcription II: The immunoglobulin mu heavy chain gene." Immunity 2(5): 427-38.

Feeney, A. J. (1990). "Lack of N regions in fetal and neonatal mouse immunoglobulin V-D-J junctional sequences." J Exp Med 172(5): 1377-90.

Fehling, H. J., A. Krotkova, C. Saint-Ruf and H. von Boehmer (1995). "Crucial role of the pre-T-cell receptor alpha gene in development of alpha beta but not gamma delta T cells." Nature 375(6534): 795-8.

Fontenot, J. D., M. A. Gavin and A. Y. Rudensky (2003). "Foxp3 programs the development and function of CD4+CD25+ regulatory T cells." Nat Immunol 4(4): 330-6.

Frasca, D., D. Nguyen, R. L. Riley and B. B. Blomberg (2003). "Decreased E12 and/or E47 transcription factor activity in the bone marrow as well as in the spleen of aged mice." Journal of immunology (Baltimore, Md. 170(2): 719-26.

Gauthier, L., B. Rossi, F. Roux, E. Termine and C. Schiff (2002). "Galectin-1 is a stromal cell ligand of the pre-B cell receptor (BCR) implicated in synapse formation between pre-B and stromal cells and in pre-BCR triggering." Proceedings of the National Academy of Sciences of the United States of America 99(20): 13014-9.

Georgopoulos, K., M. Bigby, J. H. Wang, A. Molnar, P. Wu, S. Winandy and A. Sharpe (1994). "The Ikaros gene is required for the development of all lymphoid lineages." Cell 79(1): 143-56.

Germain, R. N. and L. R. Hendrix (1991). "MHC class II structure, occupancy and surface expression determined by post-endoplasmic reticulum antigen binding." Nature 353(6340): 134-9.

Godfrey, D. I., J. Kennedy, P. Mombaerts, S. Tonegawa and A. Zlotnik (1994). "Onset of TCR-beta gene rearrangement and role of TCR-beta expression during CD3-CD4-CD8- thymocyte differentiation." J Immunol 152(10): 4783-92.

Godfrey, D. I., J. Kennedy, T. Suda and A. Zlotnik (1993). "A developmental pathway involving four phenotypically and functionally distinct subsets of CD3-CD4-CD8- triple-negative adult mouse thymocytes defined by CD44 and CD25 expression." J Immunol 150(10): 4244-52.

Godfrey, D. I., A. Zlotnik and T. Suda (1992). "Phenotypic and functional characterization of c-kit expression during intrathymic T cell development." J Immunol 149(7): 2281-5.

Gonda, H., M. Sugai, Y. Nambu, T. Katakai, Y. Agata, K. J. Mori, Y. Yokota and A. Shimizu (2003). "The balance between Pax5 and Id2 activities is the key to AID gene expression." The Journal of experimental medicine 198(9): 1427-37.

Gong, S., M. Sanchez and M. C. Nussenzweig (1996). "Counterselection against D mu is mediated through immunoglobulin (Ig)alpha-Igbeta." J Exp Med 184(6): 2079-84.

Goodnow, C. C. (1996). "Balancing immunity and tolerance: deleting and tuning lymphocyte repertoires." Proc Natl Acad Sci U S A 93(6): 2264-71.

57

Groettrup, M., K. Ungewiss, O. Azogui, R. Palacios, M. J. Owen, A. C. Hayday and H. von Boehmer (1993). "A novel disulfide-linked heterodimer on pre-T cells consists of the T cell receptor beta chain and a 33 kd glycoprotein." Cell 75(2): 283-94.

Gross, J. A., S. R. Dillon, S. Mudri, J. Johnston, A. Littau, R. Roque, M. Rixon, O. Schou, K. P. Foley, H. Haugen, S. McMillen, K. Waggie, R. W. Schreckhise, K. Shoemaker, T. Vu, M. Moore, A. Grossman and C. H. Clegg (2001). "TACI-Ig neutralizes molecules critical for B cell development and autoimmune disease. impaired B cell maturation in mice lacking BLyS." Immunity 15(2): 289-302.

Gu, H., I. Forster and K. Rajewsky (1990). "Sequence homologies, N sequence insertion and JH gene utilization in VHDJH joining: implications for the joining mechanism and the ontogenetic timing of Ly1 B cell and B-CLL progenitor generation." Embo J 9(7): 2133-40.

Gu, H., D. Kitamura and K. Rajewsky (1991). "B cell development regulated by gene rearrangement: arrest of maturation by membrane-bound D mu protein and selection of DH element reading frames." Cell 65(1): 47-54.

Guillemot, F. (1999). "Vertebrate bHLH genes and the determination of neuronal fates." Experimental cell research 253(2): 357-64.

Guinamard, R., M. Okigaki, J. Schlessinger and J. V. Ravetch (2000). "Absence of marginal zone B cells in Pyk-2-deficient mice defines their role in the humoral response." Nature immunology 1(1): 31-6.

Guo, B., R. M. Kato, M. Garcia-Lloret, M. I. Wahl and D. J. Rawlings (2000). "Engagement of the human pre-B cell receptor generates a lipid raft-dependent calcium signaling complex." Immunity 13(2): 243-53.

Haasner, D., A. Rolink and F. Melchers (1994). "Influence of surrogate L chain on DHJH-reading frame 2 suppression in mouse precursor B cells." Int Immunol 6(1): 21-30.

Haks, M. C., M. A. Oosterwegel, B. Blom, H. M. Spits and A. M. Kruisbeek (1999). "Cell-fate decisions in early T cell development: regulation by cytokine receptors and the pre-TCR." Semin Immunol 11(1): 23-37.

Han, S., K. Hathcock, B. Zheng, T. B. Kepler, R. Hodes and G. Kelsoe (1995). "Cellular interaction in germinal centers. Roles of CD40 ligand and B7-2 in established germinal centers." Journal of immunology (Baltimore, Md. 155(2): 556-67.

Hao, Z. and K. Rajewsky (2001). "Homeostasis of peripheral B cells in the absence of B cell influx from the bone marrow." J Exp Med 194(8): 1151-64.

Hardy, R. R., C. E. Carmack, S. A. Shinton, J. D. Kemp and K. Hayakawa (1991). "Resolution and characterization of pro-B and pre-pro-B cell stages in normal mouse bone marrow." J Exp Med 173(5): 1213-25.

Harker, N., T. Naito, M. Cortes, A. Hostert, S. Hirschberg, M. Tolaini, K. Roderick, K. Georgopoulos and D. Kioussis (2002). "The CD8alpha gene locus is regulated by the Ikaros family of proteins." Molecular cell 10(6): 1403-15.

Hartley, S. B., M. P. Cooke, D. A. Fulcher, A. W. Harris, S. Cory, A. Basten and C. C. Goodnow (1993). "Elimination of self-reactive B lymphocytes proceeds in two stages: arrested development and cell death." Cell 72(3): 325-35.

Hayakawa, K., R. R. Hardy, D. R. Parks and L. A. Herzenberg (1983). "The "Ly-1 B" cell subpopulation in normal immunodefective, and autoimmune mice." J Exp Med 157(1): 202-18.

58

Hayakawa, K., R. R. Hardy, A. M. Stall, L. A. Herzenberg and L. A. Herzenberg (1986). "Immunoglobulin-bearing B cells reconstitute and maintain the murine Ly-1 B cell lineage." Eur J Immunol 16(10): 1313-6.

Hayashi, K., M. Yamamoto, T. Nojima, R. Goitsuka and D. Kitamura (2003). "Distinct signaling requirements for Dmu selection, IgH allelic exclusion, pre-B cell transition, and tumor suppression in B cell progenitors." Immunity 18(6): 825-36.

Hedrick, S. M., D. I. Cohen, E. A. Nielsen and M. M. Davis (1984). "Isolation of cDNA clones encoding T cell-specific membrane-associated proteins." Nature 308(5955): 149-53.

Heemskerk, M. H., B. Blom, G. Nolan, A. P. Stegmann, A. Q. Bakker, K. Weijer, P. C. Res and H. Spits (1997). "Inhibition of T cell and promotion of natural killer cell development by the dominant negative helix loop helix factor Id3." The Journal of experimental medicine 186(9): 1597-602.

Henderson, A. and K. Calame (1998). "Transcriptional regulation during B cell development." Annual review of immunology 16: 163-200.

Henthorn, P., M. Kiledjian and T. Kadesch (1990). "Two distinct transcription factors that bind the immunoglobulin enhancer microE5/kappa 2 motif." Science 247(4941): 467-70.

Herblot, S., P. D. Aplan and T. Hoang (2002). "Gradient of E2A activity in B-cell development." Mol Cell Biol 22(3): 886-900.

Herblot, S., A. M. Steff, P. Hugo, P. D. Aplan and T. Hoang (2000). "SCL and LMO1 alter thymocyte differentiation: inhibition of E2A-HEB function and pre-T alpha chain expression." Nat Immunol 1(2): 138-44.

Hesslein, D. G., D. L. Pflugh, D. Chowdhury, A. L. Bothwell, R. Sen and D. G. Schatz (2003). "Pax5 is required for recombination of transcribed, acetylated, 5' IgH V gene segments." Genes & development 17(1): 37-42.

Ho, I. C., L. H. Yang, G. Morle and J. M. Leiden (1989). "A T-cell-specific transcriptional enhancer element 3' of C alpha in the human T-cell receptor alpha locus." Proc Natl Acad Sci U S A 86(17): 6714-8.

Hoffman, E. S., L. Passoni, T. Crompton, T. M. Leu, D. G. Schatz, A. Koff, M. J. Owen and A. C. Hayday (1996). "Productive T-cell receptor beta-chain gene rearrangement: coincident regulation of cell cycle and clonality during development in vivo." Genes Dev 10(8): 948-62.

Holmes, M. L., S. Carotta, L. M. Corcoran and S. L. Nutt (2006). "Repression of Flt3 by Pax5 is crucial for B-cell lineage commitment." Genes & development 20(8): 933-8.

Hombach, J., T. Tsubata, L. Leclercq, H. Stappert and M. Reth (1990). "Molecular components of the B-cell antigen receptor complex of the IgM class." Nature 343(6260): 760-2.

Horcher, M., A. Souabni and M. Busslinger (2001). "Pax5/BSAP maintains the identity of B cells in late B lymphopoiesis." Immunity 14(6): 779-90.

Hori, S., T. Nomura and S. Sakaguchi (2003). "Control of regulatory T cell development by the transcription factor Foxp3." Science 299(5609): 1057-61.

Horne, M. C., P. E. Roth and A. L. DeFranco (1996). "Assembly of the truncated immunoglobulin heavy chain D mu into antigen receptor-like complexes in pre-B cells but not in B cells." Immunity 4(2): 145-58.

59

Hsu, L. Y., J. Lauring, H. E. Liang, S. Greenbaum, D. Cado, Y. Zhuang and M. S. Schlissel (2003). "A conserved transcriptional enhancer regulates RAG gene expression in developing B cells." Immunity 19(1): 105-17.

Hu, J. S., E. N. Olson and R. E. Kingston (1992). "HEB, a helix-loop-helix protein related to E2A and ITF2 that can modulate the DNA-binding ability of myogenic regulatory factors." Mol Cell Biol 12(3): 1031-42.

Huang, Z., L. Nie, M. Xu and X. H. Sun (2004). "Notch-induced E2A degradation requires CHIP and Hsc70 as novel facilitators of ubiquitination." Molecular and cellular biology 24(20): 8951-62.

Ichihara, Y., H. Hayashida, S. Miyazawa and Y. Kurosawa (1989). "Only DFL16, DSP2, and DQ52 gene families exist in mouse immunoglobulin heavy chain diversity gene loci, of which DFL16 and DSP2 originate from the same primordial DH gene." Eur J Immunol 19(10): 1849-54.

Iglesias, A., M. Kopf, G. S. Williams, B. Buhler and G. Kohler (1991). "Molecular requirements for the mu-induced light chain gene rearrangement in pre-B cells." Embo J 10(8): 2147-55.

Ikawa, T., H. Kawamoto, L. Y. Wright and C. Murre (2004). "Long-term cultured E2A-deficient hematopoietic progenitor cells are pluripotent." Immunity 20(3): 349-60.

Jaleco, A. C., A. P. Stegmann, M. H. Heemskerk, F. Couwenberg, A. Q. Bakker, K. Weijer and H. Spits (1999). "Genetic modification of human B-cell development: B-cell development is inhibited by the dominant negative helix loop helix factor Id3." Blood 94(8): 2637-46.

Jameson, S. C. and M. J. Bevan (1998). "T-cell selection." Curr Opin Immunol 10(2): 214-9.

Kang, J., A. Volkmann and D. H. Raulet (2001). "Evidence that gammadelta versus alphabeta T cell fate determination is initiated independently of T cell receptor signaling." J Exp Med 193(6): 689-98.

Karasuyama, H., A. Kudo and F. Melchers (1990). "The proteins encoded by the VpreB and lambda 5 pre-B cell-specific genes can associate with each other and with mu heavy chain." J Exp Med 172(3): 969-72.

Kataoka, T., T. Kawakami, N. Takahashi and T. Honjo (1980). "Rearrangement of immunoglobulin gamma 1-chain gene and mechanism for heavy-chain class switch." Proc Natl Acad Sci U S A 77(2): 919-23.

Katz, D. H., T. Hamaoka, M. E. Dorf and B. Benacerraf (1973). "Cell interactions between histoincompatible T and B lymphocytes. The H-2 gene complex determines successful physiologic lymphocyte interactions." Proc Natl Acad Sci U S A 70(9): 2624-8.

Kaufman, J. F., C. Auffray, A. J. Korman, D. A. Shackelford and J. Strominger (1984). "The class II molecules of the human and murine major histocompatibility complex." Cell 36(1): 1-13.

Kawabe, T., T. Naka, K. Yoshida, T. Tanaka, H. Fujiwara, S. Suematsu, N. Yoshida, T. Kishimoto and H. Kikutani (1994). "The immune responses in CD40-deficient mice: impaired immunoglobulin class switching and germinal center formation." Immunity 1(3): 167-78.

Kee, B. L. and C. Murre (1998). "Induction of early B cell factor (EBF) and multiple B lineage genes by the basic helix-loop-helix transcription factor E12." J Exp Med 188(4): 699-713.

Kee, B. L. and C. Murre (2001). "Transcription factor regulation of B lineage commitment." Curr Opin Immunol 13(2): 180-5.

Kee, B. L., M. W. Quong and C. Murre (2000). "E2A proteins: essential regulators at multiple stages of B-cell development." Immunol Rev 175: 138-49.

60

Kee, B. L., R. R. Rivera and C. Murre (2001). "Id3 inhibits B lymphocyte progenitor growth and survival in response to TGF-beta." Nat Immunol 2(3): 242-7.

Khattri, R., T. Cox, S. A. Yasayko and F. Ramsdell (2003). "An essential role for Scurfin in CD4+CD25+ T regulatory cells." Nat Immunol 4(4): 337-42.

Kim, D., X. C. Peng and X. H. Sun (1999). "Massive apoptosis of thymocytes in T-cell-deficient Id1 transgenic mice." Mol Cell Biol 19(12): 8240-53.

Kisielow, P., H. Bluthmann, U. D. Staerz, M. Steinmetz and H. von Boehmer (1988). "Tolerance in T-cell-receptor transgenic mice involves deletion of nonmature CD4+8+ thymocytes." Nature 333(6175): 742-6.

Kitamura, D., A. Kudo, S. Schaal, W. Muller, F. Melchers and K. Rajewsky (1992). "A critical role of lambda 5 protein in B cell development." Cell 69(5): 823-31.

Kitamura, D., J. Roes, R. Kuhn and K. Rajewsky (1991). "A B cell-deficient mouse by targeted disruption of the membrane exon of the immunoglobulin mu chain gene." Nature 350(6317): 423-6.

Kraus, M., L. I. Pao, A. Reichlin, Y. Hu, B. Canono, J. C. Cambier, M. C. Nussenzweig and K. Rajewsky (2001). "Interference with immunoglobulin (Ig)alpha immunoreceptor tyrosine-based activation motif (ITAM) phosphorylation modulates or blocks B cell development, depending on the availability of an Igbeta cytoplasmic tail." The Journal of experimental medicine 194(4): 455-69.

Kraus, M., K. Saijo, R. M. Torres and K. Rajewsky (1999). "Ig-alpha cytoplasmic truncation renders immature B cells more sensitive to antigen contact." Immunity 11(5): 537-45.

Kudo, A. and F. Melchers (1987). "A second gene, VpreB in the lambda 5 locus of the mouse, which appears to be selectively expressed in pre-B lymphocytes." Embo J 6(8): 2267-72.

Kumararatne, D. S. and I. C. MacLennan (1981). "Cells of the marginal zone of the spleen are lymphocytes derived from recirculating precursors." Eur J Immunol 11(11): 865-9.

Kuo, C. T. and J. M. Leiden (1999). "Transcriptional regulation of T lymphocyte development and function." Annual review of immunology 17: 149-87.

Kurosawa, Y. and S. Tonegawa (1982). "Organization, structure, and assembly of immunoglobulin heavy chain diversity DNA segments." J Exp Med 155(1): 201-18.

Lam, K. P., R. Kuhn and K. Rajewsky (1997). "In vivo ablation of surface immunoglobulin on mature B cells by inducible gene targeting results in rapid cell death." Cell 90(6): 1073-83.

Langlands, K., X. Yin, G. Anand and E. V. Prochownik (1997). "Differential interactions of Id proteins with basic-helix-loop-helix transcription factors." J Biol Chem 272(32): 19785-93.

Lassar, A. B., R. L. Davis, W. E. Wright, T. Kadesch, C. Murre, A. Voronova, D. Baltimore and H. Weintraub (1991). "Functional activity of myogenic HLH proteins requires hetero-oligomerization with E12/E47-like proteins in vivo." Cell 66(2): 305-15.

Leadbetter, E. A., I. R. Rifkin, A. M. Hohlbaum, B. C. Beaudette, M. J. Shlomchik and A. Marshak-Rothstein (2002). "Chromatin-IgG complexes activate B cells by dual engagement of IgM and Toll-like receptors." Nature 416(6881): 603-7.

Ledent, V. and M. Vervoort (2001). "The basic helix-loop-helix protein family: comparative genomics and phylogenetic analysis." Genome research 11(5): 754-70.

61

Levelt, C. N., P. Mombaerts, A. Iglesias, S. Tonegawa and K. Eichmann (1993). "Restoration of early thymocyte differentiation in T-cell receptor beta-chain-deficient mutant mice by transmembrane signaling through CD3 epsilon." Proc Natl Acad Sci U S A 90(23): 11401-5.

Lin, H. and R. Grosschedl (1995). "Failure of B-cell differentiation in mice lacking the transcription factor EBF." Nature 376(6537): 263-7.

Littlewood, T., Evan, G.I. (1998). "Helix-loop-helix transcription factors." Oxford: Oxford University Press.

Liu, X., A. Adams, K. F. Wildt, B. Aronow, L. Feigenbaum and R. Bosselut (2003). "Restricting Zap70 expression to CD4+CD8+ thymocytes reveals a T cell receptor-dependent proofreading mechanism controlling the completion of positive selection." J Exp Med 197(3): 363-73.

Liu, X. and R. Bosselut (2004). "Duration of TCR signaling controls CD4-CD8 lineage differentiation in vivo." Nat Immunol 5(3): 280-8.

Livant, D., C. Blatt and L. Hood (1986). "One heavy chain variable region gene segment subfamily in the BALB/c mouse contains 500-1000 or more members." Cell 47(3): 461-70.

Loder, F., B. Mutschler, R. J. Ray, C. J. Paige, P. Sideras, R. Torres, M. C. Lamers and R. Carsetti (1999). "B cell development in the spleen takes place in discrete steps and is determined by the quality of B cell receptor-derived signals." The Journal of experimental medicine 190(1): 75-89.

Loffert, D., A. Ehlich, W. Muller and K. Rajewsky (1996). "Surrogate light chain expression is required to establish immunoglobulin heavy chain allelic exclusion during early B cell development." Immunity 4(2): 133-44.

Lucas, J. A., L. O. Atherly and L. J. Berg (2002). "The absence of Itk inhibits positive selection without changing lineage commitment." J Immunol 168(12): 6142-51.

Mackarehtschian, K., J. D. Hardin, K. A. Moore, S. Boast, S. P. Goff and I. R. Lemischka (1995). "Targeted disruption of the flk2/flt3 gene leads to deficiencies in primitive hematopoietic progenitors." Immunity 3(1): 147-61.

Maier, H., R. Ostraat, H. Gao, S. Fields, S. A. Shinton, K. L. Medina, T. Ikawa, C. Murre, H. Singh, R. R. Hardy and J. Hagman (2004). "Early B cell factor cooperates with Runx1 and mediates epigenetic changes associated with mb-1 transcription." Nature immunology 5(10): 1069-77.

Maillard, I., S. H. Adler and W. S. Pear (2003). "Notch and the immune system." Immunity 19(6): 781-91.

Maki, K., S. Sunaga, Y. Komagata, Y. Kodaira, A. Mabuchi, H. Karasuyama, K. Yokomuro, J. I. Miyazaki and K. Ikuta (1996). "Interleukin 7 receptor-deficient mice lack gammadelta T cells." Proc Natl Acad Sci U S A 93(14): 7172-7.

Malissen, M., A. Gillet, L. Ardouin, G. Bouvier, J. Trucy, P. Ferrier, E. Vivier and B. Malissen (1995). "Altered T cell development in mice with a targeted mutation of the CD3-epsilon gene." Embo J 14(19): 4641-53.

Mallick, C. A., E. C. Dudley, J. L. Viney, M. J. Owen and A. C. Hayday (1993). "Rearrangement and diversity of T cell receptor beta chain genes in thymocytes: a critical role for the beta chain in development." Cell 73(3): 513-9.

Malynn, B. A., A. C. Shaw, F. Young, V. Stewart and F. W. Alt (2002). "Truncated immunoglobulin Dmu causes incomplete developmental progression of RAG-deficient pro-B cells." Mol Immunol 38(7): 547-56.

62

Manz, J., K. Denis, O. Witte, R. Brinster and U. Storb (1988). "Feedback inhibition of immunoglobulin gene rearrangement by membrane mu, but not by secreted mu heavy chains." J Exp Med 168(4): 1363-81.

Maraskovsky, E., J. J. Peschon, H. McKenna, M. Teepe and A. Strasser (1998). "Overexpression of Bcl-2 does not rescue impaired B lymphopoiesis in IL-7 receptor-deficient mice but can enhance survival of mature B cells." Int Immunol 10(9): 1367-75.

Martensson, I. L., A. Rolink, F. Melchers, C. Mundt, S. Licence and T. Shimizu (2002). "The pre-B cell receptor and its role in proliferation and Ig heavy chain allelic exclusion." Semin Immunol 14(5): 335-42.

Martin, F. and J. F. Kearney (2002). "Marginal-zone B cells." Nat Rev Immunol 2(5): 323-35.

Massari, M. E., P. A. Grant, M. G. Pray-Grant, S. L. Berger, J. L. Workman and C. Murre (1999). "A conserved motif present in a class of helix-loop-helix proteins activates transcription by direct recruitment of the SAGA complex." Molecular cell 4(1): 63-73.

Massari, M. E. and C. Murre (2000). "Helix-loop-helix proteins: regulators of transcription in eucaryotic organisms." Molecular and cellular biology 20(2): 429-40.

Matechak, E. O., N. Killeen, S. M. Hedrick and B. J. Fowlkes (1996). "MHC class II-specific T cells can develop in the CD8 lineage when CD4 is absent." Immunity 4(4): 337-47.

McKercher, S. R., B. E. Torbett, K. L. Anderson, G. W. Henkel, D. J. Vestal, H. Baribault, M. Klemsz, A. J. Feeney, G. E. Wu, C. J. Paige and R. A. Maki (1996). "Targeted disruption of the PU.1 gene results in multiple hematopoietic abnormalities." The EMBO journal 15(20): 5647-58.

Medina, K. L., J. M. Pongubala, K. L. Reddy, D. W. Lancki, R. Dekoter, M. Kieslinger, R. Grosschedl and H. Singh (2004). "Assembling a gene regulatory network for specification of the B cell fate." Developmental cell 7(4): 607-17.

Medzhitov, R. (2001). "Toll-like receptors and innate immunity." Nat Rev Immunol 1(2): 135-45.

Meek, K. (1990). "Analysis of junctional diversity during B lymphocyte development." Science 250(4982): 820-3.

Melamed, D., R. J. Benschop, J. C. Cambier and D. Nemazee (1998). "Developmental regulation of B lymphocyte immune tolerance compartmentalizes clonal selection from receptor selection." Cell 92(2): 173-82.

Melchers, F. (2005). "The pre-B-cell receptor: selector of fitting immunoglobulin heavy chains for the B-cell repertoire." Nat Rev Immunol 5(7): 578-84.

Mikkola, I., B. Heavey, M. Horcher and M. Busslinger (2002). "Reversion of B cell commitment upon loss of Pax5 expression." Science 297(5578): 110-3.

Molina, T. J., K. Kishihara, D. P. Siderovski, W. van Ewijk, A. Narendran, E. Timms, A. Wakeham, C. J. Paige, K. U. Hartmann, A. Veillette and et al. (1992). "Profound block in thymocyte development in mice lacking p56lck." Nature 357(6374): 161-4.

Mombaerts, P., A. R. Clarke, M. A. Rudnicki, J. Iacomini, S. Itohara, J. J. Lafaille, L. Wang, Y. Ichikawa, R. Jaenisch, M. L. Hooper and et al. (1992a). "Mutations in T-cell antigen receptor genes alpha and beta block thymocyte development at different stages." Nature 360(6401): 225-31.

Mombaerts, P., J. Iacomini, R. S. Johnson, K. Herrup, S. Tonegawa and V. E. Papaioannou (1992b). "RAG-1-deficient mice have no mature B and T lymphocytes." Cell 68(5): 869-77.

63

Morgenstern, B. and W. R. Atchley (1999). "Evolution of bHLH transcription factors: modular evolution by domain shuffling?" Molecular biology and evolution 16(12): 1654-63.

Morrison, L. A., A. E. Lukacher, V. L. Braciale, D. P. Fan and T. J. Braciale (1986). "Differences in antigen presentation to MHC class I-and class II-restricted influenza virus-specific cytolytic T lymphocyte clones." J Exp Med 163(4): 903-21.

Morrow, M. A., G. Lee, S. Gillis, G. D. Yancopoulos and F. W. Alt (1992). "Interleukin-7 induces N-myc and c-myc expression in normal precursor B lymphocytes." Genes Dev 6(1): 61-70.

Mosmann, T. R., H. Cherwinski, M. W. Bond, M. A. Giedlin and R. L. Coffman (1986). "Two types of murine helper T cell clone. I. Definition according to profiles of lymphokine activities and secreted proteins." J Immunol 136(7): 2348-57.

Muljo, S. A. and M. S. Schlissel (2002). "The variable, C(H)1, C(H)2 and C(H)3 domains of Ig heavy chain are dispensable for pre-BCR function in transgenic mice." International immunology 14(6): 577-84.

Mundt, C., S. Licence, T. Shimizu, F. Melchers and I. L. Martensson (2001). "Loss of precursor B cell expansion but not allelic exclusion in VpreB1/VpreB2 double-deficient mice." J Exp Med 193(4): 435-45.

Murre, C., Baltimore, D. (1992). "The helix-loop-helix motif: structure and function." Transcriptional regulation: 861-79.

Murre, C., P. S. McCaw and D. Baltimore (1989a). "A new DNA binding and dimerization motif in immunoglobulin enhancer binding, daughterless, MyoD, and myc proteins." Cell 56(5): 777-83.

Murre, C., P. S. McCaw, H. Vaessin, M. Caudy, L. Y. Jan, Y. N. Jan, C. V. Cabrera, J. N. Buskin, S. D. Hauschka, A. B. Lassar and et al. (1989b). "Interactions between heterologous helix-loop-helix proteins generate complexes that bind specifically to a common DNA sequence." Cell 58(3): 537-44.

Murre, C., A. Voronova and D. Baltimore (1991). "B-cell- and myocyte-specific E2-box-binding factors contain E12/E47-like subunits." Mol Cell Biol 11(2): 1156-60.

Namen, A. E., S. Lupton, K. Hjerrild, J. Wignall, D. Y. Mochizuki, A. Schmierer, B. Mosley, C. J. March, D. Urdal and S. Gillis (1988). "Stimulation of B-cell progenitors by cloned murine interleukin-7." Nature 333(6173): 571-3.

Nichogiannopoulou, A., M. Trevisan, S. Neben, C. Friedrich and K. Georgopoulos (1999). "Defects in hemopoietic stem cell activity in Ikaros mutant mice." The Journal of experimental medicine 190(9): 1201-14.

Nie, L., M. Xu, A. Vladimirova and X. H. Sun (2003). "Notch-induced E2A ubiquitination and degradation are controlled by MAP kinase activities." The EMBO journal 22(21): 5780-92.

Norment, A. M., R. D. Salter, P. Parham, V. H. Engelhard and D. R. Littman (1988). "Cell-cell adhesion mediated by CD8 and MHC class I molecules." Nature 336(6194): 79-81.

Nossal, G. J., A. Szenberg, G. L. Ada and C. M. Austin (1964). "Single Cell Studies On 19s Antibody Production." J Exp Med 119: 485-502.

Nussenzweig, M. C., A. C. Shaw, E. Sinn, D. B. Danner, K. L. Holmes, H. C. Morse, 3rd and P. Leder (1987). "Allelic exclusion in transgenic mice that express the membrane form of immunoglobulin mu." Science 236(4803): 816-9.

64

Nutt, S. L., B. Heavey, A. G. Rolink and M. Busslinger (1999). "Commitment to the B-lymphoid lineage depends on the transcription factor Pax5." Nature 401(6753): 556-62.

Nutt, S. L., A. M. Morrison, P. Dorfler, A. Rolink and M. Busslinger (1998). "Identification of BSAP (Pax-5) target genes in early B-cell development by loss- and gain-of-function experiments." The EMBO journal 17(8): 2319-33.

O'Riordan, M. and R. Grosschedl (1999). "Coordinate regulation of B cell differentiation by the transcription factors EBF and E2A." Immunity 11(1): 21-31.

Oettinger, M. A., D. G. Schatz, C. Gorka and D. Baltimore (1990). "RAG-1 and RAG-2, adjacent genes that synergistically activate V(D)J recombination." Science 248(4962): 1517-23.

Okamura, R. M., M. Sigvardsson, J. Galceran, S. Verbeek, H. Clevers and R. Grosschedl (1998). "Redundant regulation of T cell differentiation and TCRalpha gene expression by the transcription factors LEF-1 and TCF-1." Immunity 8(1): 11-20.

Ordentlich, P., A. Lin, C. P. Shen, C. Blaumueller, K. Matsuno, S. Artavanis-Tsakonas and T. Kadesch (1998). "Notch inhibition of E47 supports the existence of a novel signaling pathway." Molecular and cellular biology 18(4): 2230-9.

Osmond, D. G. and G. J. Nossal (1974). "Differentiation of lymphocytes in mouse bone marrow. II. Kinetics of maturation and renewal of antiglobulin-binding cells studied by double labeling." Cell Immunol 13(1): 132-45.

Pan, L., S. Sato, J. P. Frederick, X. H. Sun and Y. Zhuang (1999). "Impaired immune responses and B-cell proliferation in mice lacking the Id3 gene." Molecular and cellular biology 19(9): 5969-80.

Pappu, R., A. M. Cheng, B. Li, Q. Gong, C. Chiu, N. Griffin, M. White, B. P. Sleckman and A. C. Chan (1999). "Requirement for B cell linker protein (BLNK) in B cell development." Science 286(5446): 1949-54.

Park, S. T., G. P. Nolan and X. H. Sun (1999). "Growth inhibition and apoptosis due to restoration of E2A activity in T cell acute lymphoblastic leukemia cells." The Journal of experimental medicine 189(3): 501-8.

Parker, M. J., S. Licence, L. Erlandsson, G. R. Galler, L. Chakalova, C. S. Osborne, G. Morgan, P. Fraser, H. Jumaa, T. H. Winkler, J. Skok and I. L. Martensson (2005). "The pre-B-cell receptor induces silencing of VpreB and lambda5 transcription." Embo J 24(22): 3895-905.

Pearce, E. L., D. J. Shedlock and H. Shen (2004). "Functional characterization of MHC class II-restricted CD8+CD4- and CD8-CD4- T cell responses to infection in CD4-/- mice." J Immunol 173(4): 2494-9.

Perlmutter, R. M., J. F. Kearney, S. P. Chang and L. E. Hood (1985). "Developmentally controlled expression of immunoglobulin VH genes." Science 227(4694): 1597-601.

Peschon, J. J., P. J. Morrissey, K. H. Grabstein, F. J. Ramsdell, E. Maraskovsky, B. C. Gliniak, L. S. Park, S. F. Ziegler, D. E. Williams, C. B. Ware, J. D. Meyer and B. L. Davison (1994). "Early lymphocyte expansion is severely impaired in interleukin 7 receptor-deficient mice." J Exp Med 180(5): 1955-60.

Petersson, K., F. Ivars and M. Sigvardsson (2002). "The pT alpha promoter and enhancer are direct targets for transactivation by E box-binding proteins." Eur J Immunol 32(3): 911-20.

Petrie, H. T., F. Livak, D. Burtrum and S. Mazel (1995). "T cell receptor gene recombination patterns and mechanisms: cell death, rescue, and T cell production." J Exp Med 182(1): 121-7.

65

Polli, M., A. Dakic, A. Light, L. Wu, D. M. Tarlinton and S. L. Nutt (2005). "The development of functional B lymphocytes in conditional PU.1 knock-out mice." Blood 106(6): 2083-90.

Porcher, C., W. Swat, K. Rockwell, Y. Fujiwara, F. W. Alt and S. H. Orkin (1996). "The T cell leukemia oncoprotein SCL/tal-1 is essential for development of all hematopoietic lineages." Cell 86(1): 47-57.

Pui, J. C., D. Allman, L. Xu, S. DeRocco, F. G. Karnell, S. Bakkour, J. Y. Lee, T. Kadesch, R. R. Hardy, J. C. Aster and W. S. Pear (1999). "Notch1 expression in early lymphopoiesis influences B versus T lineage determination." Immunity 11(3): 299-308.

Quong, M. W., D. P. Harris, S. L. Swain and C. Murre (1999). "E2A activity is induced during B-cell activation to promote immunoglobulin class switch recombination." Embo J 18(22): 6307-18.

Quong, M. W., A. Martensson, A. W. Langerak, R. R. Rivera, D. Nemazee and C. Murre (2004). "Receptor editing and marginal zone B cell development are regulated by the helix-loop-helix protein, E2A." J Exp Med 199(8): 1101-12.

Quong, M. W., M. E. Massari, R. Zwart and C. Murre (1993). "A new transcriptional-activation motif restricted to a class of helix-loop-helix proteins is functionally conserved in both yeast and mammalian cells." Molecular and cellular biology 13(2): 792-800.

Radic, M. Z. and M. Zouali (1996). "Receptor editing, immune diversification, and self-tolerance." Immunity 5(6): 505-11.

Radtke, F., A. Wilson, G. Stark, M. Bauer, J. van Meerwijk, H. R. MacDonald and M. Aguet (1999). "Deficient T cell fate specification in mice with an induced inactivation of Notch1." Immunity 10(5): 547-58.

Raff, M. C., M. Megson, J. J. Owen and M. D. Cooper (1976). "Early production of intracellular IgM by B-lymphocyte precursors in mouse." Nature 259(5540): 224-6.

Rajewsky, K. (1996). "Clonal selection and learning in the antibody system." Nature 381(6585): 751-8.

Reizis, B. and P. Leder (1999). "Expression of the mouse pre-T cell receptor alpha gene is controlled by an upstream region containing a transcriptional enhancer." J Exp Med 189(10): 1669-78.

Reizis, B. and P. Leder (2001). "The upstream enhancer is necessary and sufficient for the expression of the pre-T cell receptor alpha gene in immature T lymphocytes." J Exp Med 194(7): 979-90.

Reizis, B. and P. Leder (2002). "Direct induction of T lymphocyte-specific gene expression by the mammalian Notch signaling pathway." Genes Dev 16(3): 295-300.

Reth, M. (1989). "Antigen receptor tail clue." Nature 338(6214): 383-4.

Reth, M. G. and F. W. Alt (1984). "Novel immunoglobulin heavy chains are produced from DJH gene segment rearrangements in lymphoid cells." Nature 312(5993): 418-23.

Reth, M. G., P. Ammirati, S. Jackson and F. W. Alt (1985). "Regulated progression of a cultured pre-B-cell line to the B-cell stage." Nature 317(6035): 353-5.

Riechmann, V., I. van Cruchten and F. Sablitzky (1994). "The expression pattern of Id4, a novel dominant negative helix-loop-helix protein, is distinct from Id1, Id2 and Id3." Nucleic Acids Res 22(5): 749-55.

Rivera, R. R., C. P. Johns, J. Quan, R. S. Johnson and C. Murre (2000). "Thymocyte selection is regulated by the helix-loop-helix inhibitor protein, Id3." Immunity 12(1): 17-26.

66

Rolink, A., U. Grawunder, T. H. Winkler, H. Karasuyama and F. Melchers (1994). "IL-2 receptor alpha chain (CD25, TAC) expression defines a crucial stage in pre-B cell development." Int Immunol 6(8): 1257-64.

Rolink, A. G., S. L. Nutt, F. Melchers and M. Busslinger (1999). "Long-term in vivo reconstitution of T-cell development by Pax5-deficient B-cell progenitors." Nature 401(6753): 603-6.

Romanow, W. J., A. W. Langerak, P. Goebel, I. L. Wolvers-Tettero, J. J. van Dongen, A. J. Feeney and C. Murre (2000). "E2A and EBF act in synergy with the V(D)J recombinase to generate a diverse immunoglobulin repertoire in nonlymphoid cells." Mol Cell 5(2): 343-53.

Rosenbauer, F., K. Wagner, J. L. Kutok, H. Iwasaki, M. M. Le Beau, Y. Okuno, K. Akashi, S. Fiering and D. G. Tenen (2004). "Acute myeloid leukemia induced by graded reduction of a lineage-specific transcription factor, PU.1." Nature genetics 36(6): 624-30.

Rothenberg, E. V. (2002). "T-lineage specification and commitment: a gene regulation perspective." Seminars in immunology 14(6): 431-40.

Saarikettu, J., N. Sveshnikova and T. Grundstrom (2004). "Calcium/calmodulin inhibition of transcriptional activity of E-proteins by prevention of their binding to DNA." The Journal of biological chemistry 279(39): 41004-11.

Sabbattini, P., M. Lundgren, A. Georgiou, C. Chow, G. Warnes and N. Dillon (2001). "Binding of Ikaros to the lambda5 promoter silences transcription through a mechanism that does not require heterochromatin formation." The EMBO journal 20(11): 2812-22.

Saijo, K., C. Schmedt, I. H. Su, H. Karasuyama, C. A. Lowell, M. Reth, T. Adachi, A. Patke, A. Santana and A. Tarakhovsky (2003). "Essential role of Src-family protein tyrosine kinases in NF-kappaB activation during B cell development." Nature immunology 4(3): 274-9.

Saint-Ruf, C., K. Ungewiss, M. Groettrup, L. Bruno, H. J. Fehling and H. von Boehmer (1994). "Analysis and expression of a cloned pre-T cell receptor gene." Science 266(5188): 1208-12.

Sakaguchi, N. and F. Melchers (1986). "Lambda 5, a new light-chain-related locus selectively expressed in pre-B lymphocytes." Nature 324(6097): 579-82.

Sakaguchi, S., N. Sakaguchi, M. Asano, M. Itoh and M. Toda (1995). "Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor alpha-chains (CD25). Breakdown of a single mechanism of self-tolerance causes various autoimmune diseases." J Immunol 155(3): 1151-64.

Sakaguchi, S., N. Sakaguchi, J. Shimizu, S. Yamazaki, T. Sakihama, M. Itoh, Y. Kuniyasu, T. Nomura, M. Toda and T. Takahashi (2001). "Immunologic tolerance maintained by CD25+ CD4+ regulatory T cells: their common role in controlling autoimmunity, tumor immunity, and transplantation tolerance." Immunol Rev 182: 18-32.

Sakano, H., R. Maki, Y. Kurosawa, W. Roeder and S. Tonegawa (1980). "Two types of somatic recombination are necessary for the generation of complete immunoglobulin heavy-chain genes." Nature 286(5774): 676-83.

Salmon, P., M. Mong, X. J. Kang, D. Cado and E. Robey (1999). "The role of CD8 alpha' in the CD4 versus CD8 lineage choice." J Immunol 163(10): 5312-8.

Salomon, B., D. J. Lenschow, L. Rhee, N. Ashourian, B. Singh, A. Sharpe and J. A. Bluestone (2000). "B7/CD28 costimulation is essential for the homeostasis of the CD4+CD25+ immunoregulatory T cells that control autoimmune diabetes." Immunity 12(4): 431-40.

67

Samelson, L. E., J. B. Harford and R. D. Klausner (1985). "Identification of the components of the murine T cell antigen receptor complex." Cell 43(1): 223-31.

Sanchez, M., Z. Misulovin, A. L. Burkhardt, S. Mahajan, T. Costa, R. Franke, J. B. Bolen and M. Nussenzweig (1993). "Signal transduction by immunoglobulin is mediated through Ig alpha and Ig beta." J Exp Med 178(3): 1049-55.

Sawada, S. and D. R. Littman (1993). "A heterodimer of HEB and an E12-related protein interacts with the CD4 enhancer and regulates its activity in T-cell lines." Mol Cell Biol 13(9): 5620-8.

Sayegh, C. E., M. W. Quong, Y. Agata and C. Murre (2003). "E-proteins directly regulate expression of activation-induced deaminase in mature B cells." Nature immunology 4(6): 586-93.

Schaniel, C., L. Bruno, F. Melchers and A. G. Rolink (2002). "Multiple hematopoietic cell lineages develop in vivo from transplanted Pax5-deficient pre-B I-cell clones." Blood 99(2): 472-8.

Schatz, D. G., M. A. Oettinger and D. Baltimore (1989). "The V(D)J recombination activating gene, RAG-1." Cell 59(6): 1035-48.

Schebesta, M., P. L. Pfeffer and M. Busslinger (2002). "Control of pre-BCR signaling by Pax5-dependent activation of the BLNK gene." Immunity 17(4): 473-85.

Schiemann, B., J. L. Gommerman, K. Vora, T. G. Cachero, S. Shulga-Morskaya, M. Dobles, E. Frew and M. L. Scott (2001). "An essential role for BAFF in the normal development of B cells through a BCMA-independent pathway." Science 293(5537): 2111-4.

Schilham, M. W., M. A. Oosterwegel, P. Moerer, J. Ya, P. A. de Boer, M. van de Wetering, S. Verbeek, W. H. Lamers, A. M. Kruisbeek, A. Cumano and H. Clevers (1996). "Defects in cardiac outflow tract formation and pro-B-lymphocyte expansion in mice lacking Sox-4." Nature 380(6576): 711-4.

Schlissel, M., A. Voronova and D. Baltimore (1991). "Helix-loop-helix transcription factor E47 activates germ-line immunoglobulin heavy-chain gene transcription and rearrangement in a pre-T-cell line." Genes Dev 5(8): 1367-76.

Schweighoffer, E., L. Vanes, A. Mathiot, T. Nakamura and V. L. Tybulewicz (2003). "Unexpected requirement for ZAP-70 in pre-B cell development and allelic exclusion." Immunity 18(4): 523-33.

Scott, E. W., M. C. Simon, J. Anastasi and H. Singh (1994). "Requirement of transcription factor PU.1 in the development of multiple hematopoietic lineages." Science 265(5178): 1573-7.

Seet, C. S., R. L. Brumbaugh and B. L. Kee (2004). "Early B cell factor promotes B lymphopoiesis with reduced interleukin 7 responsiveness in the absence of E2A." J Exp Med 199(12): 1689-700.

Shaffer, A. L. and M. S. Schlissel (1997). "A truncated heavy chain protein relieves the requirement for surrogate light chains in early B cell development." Journal of immunology (Baltimore, Md. 159(3): 1265-75.

Shapiro, A. M., M. S. Schlissel, D. Baltimore and A. L. DeFranco (1993). "Stimulation of kappa light-chain gene rearrangement by the immunoglobulin mu heavy chain in a pre-B-cell line." Mol Cell Biol 13(9): 5679-90.

Shen, C. P. and T. Kadesch (1995). "B-cell-specific DNA binding by an E47 homodimer." Mol Cell Biol 15(8): 4518-24.

68

Shimizu, T., C. Mundt, S. Licence, F. Melchers and I. L. Martensson (2002). "VpreB1/VpreB2/lambda 5 triple-deficient mice show impaired B cell development but functional allelic exclusion of the IgH locus." J Immunol 168(12): 6286-93.

Shinkai, Y., S. Koyasu, K. Nakayama, K. M. Murphy, D. Y. Loh, E. L. Reinherz and F. W. Alt (1993). "Restoration of T cell development in RAG-2-deficient mice by functional TCR transgenes." Science 259(5096): 822-5.

Shinkai, Y., G. Rathbun, K. P. Lam, E. M. Oltz, V. Stewart, M. Mendelsohn, J. Charron, M. Datta, F. Young, A. M. Stall and et al. (1992). "RAG-2-deficient mice lack mature lymphocytes owing to inability to initiate V(D)J rearrangement." Cell 68(5): 855-67.

Shivdasani, R. A., E. L. Mayer and S. H. Orkin (1995). "Absence of blood formation in mice lacking the T-cell leukaemia oncoprotein tal-1/SCL." Nature 373(6513): 432-4.

Sigvardsson, M., M. O'Riordan and R. Grosschedl (1997). "EBF and E47 collaborate to induce expression of the endogenous immunoglobulin surrogate light chain genes." Immunity 7(1): 25-36.

Singh, H., K. L. Medina and J. M. Pongubala (2005). "Contingent gene regulatory networks and B cell fate specification." Proc Natl Acad Sci U S A 102(14): 4949-53.

Sitnicka, E., C. Brakebusch, I. L. Martensson, M. Svensson, W. W. Agace, M. Sigvardsson, N. Buza-Vidas, D. Bryder, C. M. Cilio, H. Ahlenius, E. Maraskovsky, J. J. Peschon and S. E. Jacobsen (2003a). "Complementary signaling through flt3 and interleukin-7 receptor alpha is indispensable for fetal and adult B cell genesis." The Journal of experimental medicine 198(10): 1495-506.

Sitnicka, E., C. Brakebusch, I. L. Martensson, M. Svensson, W. W. Agace, M. Sigvardsson, N. Buza-Vidas, D. Bryder, C. M. Cilio, H. Ahlenius, E. Maraskovsky, J. J. Peschon and S. E. Jacobsen (2003b). "Complementary signaling through flt3 and interleukin-7 receptor alpha is indispensable for fetal and adult B cell genesis." J Exp Med 198(10): 1495-506.

Sitnicka, E., D. Bryder, K. Theilgaard-Monch, N. Buza-Vidas, J. Adolfsson and S. E. Jacobsen (2002). "Key role of flt3 ligand in regulation of the common lymphoid progenitor but not in maintenance of the hematopoietic stem cell pool." Immunity 17(4): 463-72.

Siu, G., S. P. Clark, Y. Yoshikai, M. Malissen, Y. Yanagi, E. Strauss, T. W. Mak and L. Hood (1984). "The human T cell antigen receptor is encoded by variable, diversity, and joining gene segments that rearrange to generate a complete V gene." Cell 37(2): 393-401.

Sleckman, B. P., C. H. Bassing, C. G. Bardon, A. Okada, B. Khor, J. C. Bories, R. Monroe and F. W. Alt (1998). "Accessibility control of variable region gene assembly during T-cell development." Immunol Rev 165: 121-30.

Sloan, S. R., C. P. Shen, R. McCarrick-Walmsley and T. Kadesch (1996). "Phosphorylation of E47 as a potential determinant of B-cell-specific activity." Molecular and cellular biology 16(12): 6900-8.

Smider, V. and G. Chu (1997). "The end-joining reaction in V(D)J recombination." Semin Immunol 9(3): 189-97.

Souabni, A., C. Cobaleda, M. Schebesta and M. Busslinger (2002). "Pax5 promotes B lymphopoiesis and blocks T cell development by repressing Notch1." Immunity 17(6): 781-93.

Spanopoulou, E., C. A. Roman, L. M. Corcoran, M. S. Schlissel, D. P. Silver, D. Nemazee, M. C. Nussenzweig, S. A. Shinton, R. R. Hardy and D. Baltimore (1994). "Functional immunoglobulin transgenes guide ordered B-cell differentiation in Rag-1-deficient mice." Genes Dev 8(9): 1030-42.

69

Srivastava, B., W. J. Quinn, 3rd, K. Hazard, J. Erikson and D. Allman (2005). "Characterization of marginal zone B cell precursors." The Journal of experimental medicine 202(9): 1225-34.

Staudt, L. M. and M. J. Lenardo (1991). "Immunoglobulin gene transcription." Annu Rev Immunol 9: 373-98.

Steinmetz, M., K. Minard, S. Horvath, J. McNicholas, J. Srelinger, C. Wake, E. Long, B. Mach and L. Hood (1982). "A molecular map of the immune response region from the major histocompatibility complex of the mouse." Nature 300(5887): 35-42.

Stephens, L. A. and D. Mason (2000). "CD25 is a marker for CD4+ thymocytes that prevent autoimmune diabetes in rats, but peripheral T cells with this function are found in both CD25+ and CD25- subpopulations." Journal of immunology (Baltimore, Md. 165(6): 3105-10.

Su, Y. W. and H. Jumaa (2003). "LAT links the pre-BCR to calcium signaling." Immunity 19(2): 295-305.

Sun, X. H. (1994). "Constitutive expression of the Id1 gene impairs mouse B cell development." Cell 79(5): 893-900.

Sun, X. H. and D. Baltimore (1991). "An inhibitory domain of E12 transcription factor prevents DNA binding in E12 homodimers but not in E12 heterodimers." Cell 64(2): 459-70.

Sun, X. H., N. G. Copeland, N. A. Jenkins and D. Baltimore (1991). "Id proteins Id1 and Id2 selectively inhibit DNA binding by one class of helix-loop-helix proteins." Mol Cell Biol 11(11): 5603-11.

Takeuchi, A., S. Yamasaki, K. Takase, F. Nakatsu, H. Arase, M. Onodera and T. Saito (2001). "E2A and HEB activate the pre-TCR alpha promoter during immature T cell development." J Immunol 167(4): 2157-63.

Tanigaki, K., H. Han, N. Yamamoto, K. Tashiro, M. Ikegawa, K. Kuroda, A. Suzuki, T. Nakano and T. Honjo (2002). "Notch-RBP-J signaling is involved in cell fate determination of marginal zone B cells." Nature immunology 3(5): 443-50.

Tiegs, S. L., D. M. Russell and D. Nemazee (1993). "Receptor editing in self-reactive bone marrow B cells." J Exp Med 177(4): 1009-20.

Ting, C. N., M. C. Olson, K. P. Barton and J. M. Leiden (1996). "Transcription factor GATA-3 is required for development of the T-cell lineage." Nature 384(6608): 474-8.

Tomita, K., M. Hattori, E. Nakamura, S. Nakanishi, N. Minato and R. Kageyama (1999). "The bHLH gene Hes1 is essential for expansion of early T cell precursors." Genes & development 13(9): 1203-10.

Tornberg, U. C., I. Bergqvist, M. Haury and D. Holmberg (1998). "Regulation of B lymphocyte development by the truncated immunoglobulin heavy chain protein Dmu." J Exp Med 187(5): 703-9.

Townsend, A. R., F. M. Gotch and J. Davey (1985). "Cytotoxic T cells recognize fragments of the influenza nucleoprotein." Cell 42(2): 457-67.

Tremblay, M., S. Herblot, E. Lecuyer and T. Hoang (2003). "Regulation of pT alpha gene expression by a dosage of E2A, HEB, and SCL." J Biol Chem 278(15): 12680-7.

Trinh, L. A., R. Ferrini, B. S. Cobb, A. S. Weinmann, K. Hahm, P. Ernst, I. P. Garraway, M. Merkenschlager and S. T. Smale (2001). "Down-regulation of TDT transcription in CD4(+)CD8(+) thymocytes by Ikaros proteins in direct competition with an Ets activator." Genes & development 15(14): 1817-32.

70

Tsubata, T. and M. Reth (1990). "The products of pre-B cell-specific genes (lambda 5 and VpreB) and the immunoglobulin mu chain form a complex that is transported onto the cell surface." J Exp Med 172(3): 973-6.

Tsubata, T., R. Tsubata and M. Reth (1992). "Crosslinking of the cell surface immunoglobulin (mu-surrogate light chains complex) on pre-B cells induces activation of V gene rearrangements at the immunoglobulin kappa locus." Int Immunol 4(6): 637-41.

Tyznik, A. J., J. C. Sun and M. J. Bevan (2004). "The CD8 population in CD4-deficient mice is heavily contaminated with MHC class II-restricted T cells." J Exp Med 199(4): 559-65.

Urbanek, P., Z. Q. Wang, I. Fetka, E. F. Wagner and M. Busslinger (1994). "Complete block of early B cell differentiation and altered patterning of the posterior midbrain in mice lacking Pax5/BSAP." Cell 79(5): 901-12.

Van der Put, E., D. Frasca, A. M. King, B. B. Blomberg and R. L. Riley (2004). "Decreased E47 in senescent B cell precursors is stage specific and regulated posttranslationally by protein turnover." Journal of immunology (Baltimore, Md. 173(2): 818-27.

Van Eldik, L., Watterson, D.M. (1998). "Calmodulin and Signal Transduction." Academic Preee, Inc., New York.

Van Parijs, L. and A. K. Abbas (1998). "Homeostasis and self-tolerance in the immune system: turning lymphocytes off." Science 280(5361): 243-8.

Wang, J. H., A. Nichogiannopoulou, L. Wu, L. Sun, A. H. Sharpe, M. Bigby and K. Georgopoulos (1996). "Selective defects in the development of the fetal and adult lymphoid system in mice with an Ikaros null mutation." Immunity 5(6): 537-49.

Washburn, T., E. Schweighoffer, T. Gridley, D. Chang, B. J. Fowlkes, D. Cado and E. Robey (1997). "Notch activity influences the alphabeta versus gammadelta T cell lineage decision." Cell 88(6): 833-43.

Weigert, M. G., I. M. Cesari, S. J. Yonkovich and M. Cohn (1970). "Variability in the lambda light chain sequences of mouse antibody." Nature 228(5276): 1045-7.

Viglianti, G. A., C. M. Lau, T. M. Hanley, B. A. Miko, M. J. Shlomchik and A. Marshak-Rothstein (2003). "Activation of autoreactive B cells by CpG dsDNA." Immunity 19(6): 837-47.

Wilson, A., W. Held and H. R. MacDonald (1994). "Two waves of recombinase gene expression in developing thymocytes." J Exp Med 179(4): 1355-60.

Visvader, J. E., Y. Fujiwara and S. H. Orkin (1998). "Unsuspected role for the T-cell leukemia protein SCL/tal-1 in vascular development." Genes & development 12(4): 473-9.

Vitetta, E. S. and J. W. Uhr (1975). "Immunoglobulin-receptors revisited." Science 189(4207): 964-9.

Wolfer, A., A. Wilson, M. Nemir, H. R. MacDonald and F. Radtke (2002). "Inactivation of Notch1 impairs VDJbeta rearrangement and allows pre-TCR-independent survival of early alpha beta Lineage Thymocytes." Immunity 16(6): 869-79.

von Boehmer, H., I. Aifantis, J. Feinberg, O. Lechner, C. Saint-Ruf, U. Walter, J. Buer and O. Azogui (1999). "Pleiotropic changes controlled by the pre-T-cell receptor." Curr Opin Immunol 11(2): 135-42.

von Boehmer, H. and P. Kisielow (1990). "Self-nonself discrimination by T cells." Science 248(4961): 1369-73.

71

von Freeden-Jeffry, U., P. Vieira, L. A. Lucian, T. McNeil, S. E. Burdach and R. Murray (1995). "Lymphopenia in interleukin (IL)-7 gene-deleted mice identifies IL-7 as a nonredundant cytokine." J Exp Med 181(4): 1519-26.

Vosshenrich, C. A., A. Cumano, W. Muller, J. P. Di Santo and P. Vieira (2003). "Thymic stromal-derived lymphopoietin distinguishes fetal from adult B cell development." Nature immunology 4(8): 773-9.

Wu, L., M. Antica, G. R. Johnson, R. Scollay and K. Shortman (1991). "Developmental potential of the earliest precursor cells from the adult mouse thymus." J Exp Med 174(6): 1617-27.

Wu, L., C. L. Li and K. Shortman (1996). "Thymic dendritic cell precursors: relationship to the T lymphocyte lineage and phenotype of the dendritic cell progeny." J Exp Med 184(3): 903-11.

Wu, T. T. and E. A. Kabat (1970). "An analysis of the sequences of the variable regions of Bence Jones proteins and myeloma light chains and their implications for antibody complementarity." J Exp Med 132(2): 211-50.

Yamada, Y., A. J. Warren, C. Dobson, A. Forster, R. Pannell and T. H. Rabbitts (1998). "The T cell leukemia LIM protein Lmo2 is necessary for adult mouse hematopoiesis." Proceedings of the National Academy of Sciences of the United States of America 95(7): 3890-5.

Yan, W., A. Z. Young, V. C. Soares, R. Kelley, R. Benezra and Y. Zhuang (1997). "High incidence of T-cell tumors in E2A-null mice and E2A/Id1 double-knockout mice." Molecular and cellular biology 17(12): 7317-27.

Yanagi, Y., Y. Yoshikai, K. Leggett, S. P. Clark, I. Aleksander and T. W. Mak (1984). "A human T cell-specific cDNA clone encodes a protein having extensive homology to immunoglobulin chains." Nature 308(5955): 145-9.

Yancopoulos, G. D. and F. W. Alt (1986). "Regulation of the assembly and expression of variable-region genes." Annu Rev Immunol 4: 339-68.

Yancopoulos, G. D., S. V. Desiderio, M. Paskind, J. F. Kearney, D. Baltimore and F. W. Alt (1984). "Preferential utilization of the most JH-proximal VH gene segments in pre-B-cell lines." Nature 311(5988): 727-33.

Yokota, Y., A. Mansouri, S. Mori, S. Sugawara, S. Adachi, S. Nishikawa and P. Gruss (1999). "Development of peripheral lymphoid organs and natural killer cells depends on the helix-loop-helix inhibitor Id2." Nature 397(6721): 702-6.

Young, F., B. Ardman, Y. Shinkai, R. Lansford, T. K. Blackwell, M. Mendelsohn, A. Rolink, F. Melchers and F. W. Alt (1994). "Influence of immunoglobulin heavy- and light-chain expression on B-cell differentiation." Genes Dev 8(9): 1043-57.

Young, F., E. Mizoguchi, A. K. Bhan and F. W. Alt (1997). "Constitutive Bcl-2 expression during immunoglobulin heavy chain- promoted B cell differentiation expands novel precursor B cells." Immunity 6(1): 23-33.

Zhang, J., M. Kalkum, S. Yamamura, B. T. Chait and R. G. Roeder (2004). "E protein silencing by the leukemogenic AML1-ETO fusion protein." Science 305(5688): 1286-9.

Zhuang, Y., R. J. Barndt, L. Pan, R. Kelley and M. Dai (1998). "Functional replacement of the mouse E2A gene with a human HEB cDNA." Mol Cell Biol 18(6): 3340-9.

72

Zhuang, Y., P. Cheng and H. Weintraub (1996). "B-lymphocyte development is regulated by the combined dosage of three basic helix-loop-helix genes, E2A, E2-2, and HEB." Mol Cell Biol 16(6): 2898-905.

Zhuang, Y., P. Soriano and H. Weintraub (1994). "The helix-loop-helix gene E2A is required for B cell formation." Cell 79(5): 875-84.

Zinkernagel, R. M. and P. C. Doherty (1974). "Restriction of in vitro T cell-mediated cytotoxicity in lymphocytic choriomeningitis within a syngeneic or semiallogeneic system." Nature 248(450): 701-2.